Methods of expressing angiostatic protein

ABSTRACT

The present invention comprises an endothelial inhibitor and method of use therefor. The endothelial inhibitor is a protein isolated from the blood or urine that is eluted as a single peak from C4-reverse phase high performance liquid chromatography. The endothelial inhibitor is a molecule comprising a protein having a molecular weight of between approximately 38 kilodaltons and 45 kilodaltons as determined by reducing polyacrylamide gel electrophoresis and having an amino acid sequence substantially similar to that of a murine plasminogen fragment beginning at amino acid number 98 of a murine plasminogen molecule.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. Ser. No.08/326,785, filed Oct. 20, 1994, which is a continuation-in-part of U.S.Ser. No. 08/248,629, filed Apr. 26, 1994, now U.S. Pat. No. 5,639,725.

FIELD OF THE INVENTION

The present invention relates to endothelial inhibitors, calledangiostatin, which reversibly inhibit proliferation of endothelialcells. More particularly, the present invention relates to angiostatinproteins that can be isolated from body fluids such as blood or urine,or can be synthesized by recombinant, enzymatic or chemical methods. Theangiostatin is capable of inhibiting angiogenesis related diseases andmodulating angiogenic processes. In addition, the present inventionrelates to diagnostic assays and kits for angiostatin measurement, tohistochemical kits for localization of angiostatin, to DNA sequencescoding for angiostatin and molecular probes to monitor angiostatinbiosynthesis, to antibodies that are specific for the angiostatin, tothe development of peptide agonists and antagonists to the angiostatinreceptor, to anti-angiostatin receptor-specific antibody agonists andantagonists, and to cytotoxic agents linked to angiostatin peptides.

BACKGROUND OF THE INVENTION

As used herein, the term "angiogenesis" means the generation of newblood vessels into a tissue or organ. Under normal physiologicalconditions, humans or animals undergo angiogenesis only in very specificrestricted situations. For example, angiogenesis is normally observed inwound healing, fetal and embryonal development and formation of thecorpus luteum, endometrium and placenta. The term "endothelium" means athin layer of flat epithelial cells that lines serous cavities, lymphvessels, and blood vessels.

Both controlled and uncontrolled angiogenesis are thought to proceed ina similar manner. Endothelial cells and pericytes, surrounded by abasement membrane, form capillary blood vessels. Angiogenesis beginswith the erosion of the basement membrane by enzymes released byendothelial cells and leukocytes. The endothelial cells, which line thelumen of blood vessels, then protrude through the basement membrane.Angiogenic stimulants induce the endothelial cells to migrate throughthe eroded basement membrane. The migrating cells form a "sprout" offthe parent blood vessel, where the endothelial cells undergo mitosis andproliferate. The endothelial sprouts merge with each other to formcapillary loops, creating the new blood vessel.

Persistent, unregulated angiogenesis occurs in a multiplicity of diseasestates, tumor metastasis and abnormal growth by endothelial cells andsupports the pathological damage seen in these conditions. The diversepathological disease states in which unregulated angiogenesis is presenthave been grouped together as angiogenic dependent or angiogenicassociated diseases.

The hypothesis that tumor growth is angiogenesis-dependent was firstproposed in 1971. (Folkman J., Tumor angiogenesis: Therapeuticimplications., N. Engl. Jour. Med. 285:1182 1186, 1971) In its simplestterms it states: "Once tumor `take` has occurred, every increase intumor cell population must be preceded by an increase in new capillariesconverging on the tumor." Tumor `take` is currently understood toindicate a prevascular phase of tumor growth in which a population oftumor cells occupying a few cubic millimeters volume and not exceeding afew million cells, can survive on existing host microvessels. Expansionof tumor volume beyond this phase requires the induction of newcapillary blood vessels. For example, pulmonary micrometastases in theearly prevascular phase in mice would be undetectable except by highpower microscopy on histological sections.

Examples of the indirect evidence which support this concept include:

(1) The growth rate of tumors implanted in subcutaneous transparentchambers in mice is slow and linear before neovascularization, and rapidand nearly exponential after neovascularization. (Algire GH, et al.Vascular reactions of normal and malignant tumors in vivo. I. Vascularreactions of mice to wounds and to normal and neoplastic transplants. J.Natl. Cancer Inst. 6:73-85, 1945)

(2) Tumors grown in isolated perfused organs where blood vessels do notproliferate are limited to 1-2 mm3 but expand rapidly to >1000 timesthis volume when they are transplanted to mice and becomeneovascularized. (Folkman J, et al., Tumor behavior in isolated perfusedorgans: In vitro growth and metastasis of biopsy material in rabbitthyroid and canine intestinal segments. Annals of Surgery 164:491-502,1966)

(3) Tumor growth in the avascular cornea proceeds slowly and at a linearrate, but switches to exponential growth after neovascularization.(Gimbrone, M. A., Jr. et al., Tumor growth and neovascularization: Anexperimental model using the rabbit cornea. J. Natl. Cancer Institute52:41-427, 1974)

(4) Tumors suspended in the aqueous fluid of the anterior chamber of therabbit eye, remain viable, avascular and limited in size to <1 mm³. Oncethey are implanted on the iris vascular bed, they become neovascularizedand grow rapidly, reaching 16,000 times their original volume within 2weeks. (Gimbrone M A Jr., et al., Tumor dormancy in vivo by preventionof neovascularization. J. Exp. Med. 136:261-276)

(5) When tumors are implanted on the chick embryo chorioallantoicmembrane, they grow slowly during an avascular phase of >72 hours, butdo not exceed a mean diameter of 0.93+0.29 mm. Rapid tumor expansionoccurs within 24 hours after the onset of neovascularization, and by day7 these vascularized tumors reach a mean diameter of 8.0+2.5 mm.(Knighton D., Avascular and vascular phases of tumor growth in the chickembryo. British J. Cancer, 35:347-356, 1977)

(6) Vascular casts of metastases in the rabbit liver revealheterogeneity in size of the metastases, but show a relatively uniformcut-off point for the size at which vascularization is present. Tumorsare generally avascular up to 1 mm in diameter, but are neovascularizedbeyond that diameter. (Lien W., et al., The blood supply of experimentalliver metastases. II. A microcirculatory study of normal and tumorvessels of the liver with the use of perfused silicone rubber. Surgery68:334-340, 1970)

(7) In transgenic mice which develop carcinomas in the beta cells of thepancreatic islets, pre-vascular hyperplastic islets are limited in sizeto <1 mm. At 6-7 weeks of age, 4-10% of the islets becomeneovascularized, and from these islets arise large vascularized tumorsof more than 1000 times the volume of the pre-vascular islets. (FolkmanJ, et al., Induction of angiogenesis during the transition fromhyperplasia to neoplasia. Nature 339:58-61, 1989)

(8) A specific antibody against VEGF (vascular endothelial growthfactor) reduces microvessel density and causes "significant or dramatic"inhibition of growth of three human tumors which rely on VEGF as theirsole mediator of angiogenesis (in nude mice). The antibody does notinhibit growth of the tumor cells in vitro. (Kim K J, et al., Inhibitionof vascular endothelial growth factor-induced angiogenesis suppressestumor growth in vivo. Nature 362:841-844, 1993)

(9) Anti-bFGF monoclonal antibody causes 70% inhibition of growth of amouse tumor which is dependent upon secretion of bFGF as its onlymediator of angiogenesis. The antibody does not inhibit growth of thetumor cells in vitro. (Hori A, et al., Suppression of solid tumor growthby immunoneutralizing monoclonal antibody against human basic fibroblastgrowth factor. Cancer Research, 51:6180-6184, 1991)

(10) Intraperitoneal injection of bFGF enhances growth of a primarytumor and its metastases by stimulating growth of capillary endothelialcells in the tumor. The tumor cells themselves lack receptors for bFGF,and bFGF is not a mitogen for the tumors cells in vitro. (Gross J L, etal. Modulation of solid tumor growth in vivo by bFGF. Proc. Amer. Assoc.Canc. Res. 31:79, 1990)

(11 ) A specific angiogenesis inhibitor (AGM-1470) inhibits tumor growthand metastases in vivo, but is much less active in inhibiting tumor cellproliferation in vitro. It inhibits vascular endothelial cellproliferation half-maximally at 4 logs lower concentration than itinhibits tumor cell proliferation. (Ingber D, et al., Angioinhibins:Synthetic analogues of fumagillin which inhibit angiogenesis andsuppress tumor growth. Nature, 48:555-557, 1990). There is also indirectclinical evidence that tumor growth is angiogenesis dependent.

(12) Human retinoblastomas that are metastatic to the vitreous developinto avascular spheroids which are restricted to less than 1 mm³ despitethe fact that they are viable and incorporate ³ H-thymidine (whenremoved from an enucleated eye and analyzed in vitro).

(13) Carcinoma of the ovary metastasizes to the peritoneal membrane astiny avascular white seeds (1-3 mm³). These implants rarely grow largeruntil one or more of them becomes neovascularized.

(14) Intensity of neovascularization in breast cancer (Weidner N, etal., Tumor angiogenesis correlates with metastasis in invasive breastcarcinoma. N. Engl. J. Med. 324:1-8, 1991, and Weidner N, et al., Tumorangiogenesis: A new significant and independent prognostic indicator inearly-stage breast carcinoma, J Natl. Cancer Inst. 84:1875-1887, 1992)and in prostate cancer (Weidner N, Carroll P R, Flax J, Blumenfeld W,Folkman J. Tumor angiogenesis correlates with metastasis in invasiveprostate carcinoma. American Journal of Pathology, 143(2):401-409, 1993)correlates highly with risk of future metastasis.

(15) Metastasis from human cutaneous melanoma is rare prior toneovascularization. The onset of neovascularization leads to increasedthickness of the lesion and an increasing risk of metastasis.(Srivastava A, et al., The prognostic significance of tumor vascularityin intermediate thickness (0.76-4.0 mm thick) skin melanoma. Amer. J.Pathol. 133:419-423, 1988)

(16) In bladder cancer, the urinary level of an angiogenic peptide,bFGF, is a more sensitive indicator of status and extent of disease thanis cytology. (Nguyen M, et al., Elevated levels of an angiogenicpeptide, basic fibroblast growth factor, in urine of bladder cancerpatients. J. Natl. Cancer Inst. 85:241-242, 1993)

Thus, it is clear that angiogenesis plays a major role in the metastasisof a cancer. If this angiogenic activity could be repressed oreliminated, then the tumor, although present, would not grow. In thedisease state, prevention of angiogenesis could avert the damage causedby the invasion of the new microvascular system. Therapies directed atcontrol of the angiogenic processes could lead to the abrogation ormitigation of these diseases.

What is needed therefore is a composition and method which can inhibitthe unwanted growth of blood vessels, especially into tumors. Alsoneeded is a method for detecting, measuring, and localizing thecomposition. The composition should be able to overcome the activity ofendogenous growth factors in premetastatic tumors and prevent theformation of the capillaries in the tumors thereby inhibiting the growthof the tumors. The composition, fragments of the composition, andantibodies specific to the composition, should also be able to modulatethe formation of capillaries in other angiogenic processes, such aswound healing and reproduction. The composition and method forinhibiting angiogenesis should preferably be non-toxic and produce fewside effects. Also needed is a method for detecting, measuring, andlocalizing the binding sites for the composition as well as sites ofbiosynthesis of the composition. The composition and fragments of thecomposition should be capable of being conjugated to other molecules forboth radioactive and non-radioactive labeling purposes.

SUMMARY OF THE INVENTION

In accordance with the present invention, compositions and methods areprovided that are effective for modulating angiogenesis, and inhibitingunwanted angiogenesis, especially angiogenesis related to tumor growth.The present invention includes a protein, which has been named"angiostatin", defined by its ability to overcome the angiogenicactivity of endogenous growth factors such as bFGF, in vitro, and by itamino acid sequence homology and structural similarity to an internalportion of plasminogen beginning at approximately plasminogen amino acid98. Angiostatin comprises a protein having a molecular weight of betweenapproximately 38 kilodaltons and 45 kilodaltons as determined byreducing polyacrylamide gel electrophoresis and having an amino acidsequence substantially similar to that of a fragment of murineplasminogen beginning at amino acid number 98 of an intact murineplasminogen molecule (SEQ ID NO:2).

The amino acid sequence of angiostatin varies slightly between species.For example, in human angiostatin the amino acid sequence issubstantially similar to the sequence of the above described murineplasminogen fragment, although an active human angiostatin sequence maystart at either amino acid number 97 or 99 of an intact humanplasminogen amino acid sequence. Further, fragments of human plasminogenhas similar anti-angiogenic activity as shown in a mouse tumor model. Itis to be understood that the number of amino acids in the activeangiostatin molecule may vary and all amino acid sequences that haveendothelial inhibiting activity are contemplated as being included inthe present invention.

The present invention provides methods and compositions for treatingdiseases and processes mediated by undesired and uncontrolledangiogenesis by administering to a human or animal a compositioncomprising a substantially purified angiostatin or angiostatinderivative in a dosage sufficient to inhibit angiogenesis. The presentinvention is particularly useful for treating, or for repressing thegrowth of, tumors. Administration of angiostatin to a human or animalwith prevascularized metastasized tumors will prevent the growth orexpansion of those tumors.

The present invention also encompasses DNA sequences encodingangiostatin, expression vectors containing DNA sequences encodingangiostatin, and cells containing one or more expression vectorscontaining DNA sequences encoding angiostatin. The present inventionfurther encompasses gene therapy methods whereby DNA sequences encodingangiostatin are introduced into a patient to modify in vivo angiostatinlevels.

The present invention also includes diagnostic methods and kits fordetection and measurement of angiostatin in biological fluids andtissues, and for localization of angiostatin in tissues and cells. Thediagnostic method and kit can be in any configuration well known tothose of ordinary skill in the art. The present invention also includesantibodies specific for the angiostatin molecule and portions thereof,and antibodies that inhibit the binding of antibodies specific for theangiostatin. These antibodies can be polyclonal antibodies or monoclonalantibodies. The antibodies specific for the angiostatin can be used indiagnostic kits to detect the presence and quantity of angiostatin whichis diagnostic or prognostic for the occurrence or recurrence of canceror other disease mediated by angiogenesis. Antibodies specific forangiostatin may also be administered to a human or animal to passivelyimmunize the human or animal against angiostatin, thereby reducingangiogenic inhibition.

The present invention also includes diagnostic methods and kits fordetecting the presence and quantity of antibodies that bind angiostatinin body fluids. The diagnostic method and kit can be in anyconfiguration well known to those of ordinary skill in the art.

The present invention also includes anti-angiostatin receptor-specificantibodies that bind to the angiostatin receptor and transmit theappropriate signal to the cell and act as agonists or antagonists.

The present invention also includes angiostatin peptide fragments andanalogs that can be labeled isotopically or with other molecules orproteins for use in the detection and visualization of angiostatinbinding sites with techniques, including, but not limited to, positronemission tomography, autoradiography, flow cytometry, radioreceptorbinding assays, and immunohistochemistry.

These angiostatin peptides and analogs also act as agonists andantagonists at the angiostatin receptor, thereby enhancing or blockingthe biological activity of angiostatin. Such peptides are used in theisolation of the angiostatin receptor.

The present invention also includes angiostatin, angiostatin fragments,angiostatin antisera, or angiostatin receptor agonists and angiostatinreceptor antagonists linked to cytotoxic agents for therapeutic andresearch applications. Still further, angiostatin, angiostatinfragments, angiostatin antisera, angiostatin receptor agonists andangiostatin receptor antagonists are combined with pharmaceuticallyacceptable excipients, and optionally sustained-release compounds orcompositions, such as biodegradable polymers, to form therapeuticcompositions.

The present invention includes molecular probes for the ribonucleic acidand deoxyribonucleic acid involved in transcription and translation ofangiostatin. These molecular probes provide means to detect and measureangiostatin biosynthesis in tissues and cells.

Accordingly, it is an object of the present invention to provide acomposition comprising an angiostatin.

It is another object of the present invention to provide a method oftreating diseases and processes that are mediated by angiogenesis.

It is yet another object of the present invention to provide adiagnostic or prognostic method and kit for detecting the presence andamount of angiostatin in a body fluid or tissue.

It is yet another object of the present invention to provide a methodand composition for treating diseases and processes that are mediated byangiogenesis including, but not limited to, hemangioma, solid tumors,blood borne tumors, leukemia, metastasis, telangiectasia, psoriasis,scleroderma, pyogenic granuloma, myocardial angiogenesis, Crohn'sdisease, plaque neovascularization, coronary collaterals, cerebralcollaterals, arteriovenous malformations, ischemic limb angiogenesis,corneal diseases, rubeosis, neovascular glaucoma, diabetic retinopathy,retrolental fibroplasia, arthritis, diabetic neovascularization, maculardegeneration, wound healing, peptic ulcer, Helicobacter relateddiseases, fractures, keloids, vasculogenesis, hematopoiesis, ovulation,menstruation, placentation, and cat scratch fever.

It is another object of the present invention to provide a compositionfor treating or repressing the growth of a cancer.

It is an object of the present invention to provide compounds thatmodulate or mimic the production or activity of enzymes that produceangiostatin in vivo or in vitro.

It is a further object of the present invention to provide angiostatinor anti-angiostatin antibodies by direct injection of angiostatin DNAinto a human or animal needing such angiostatin or anti-angiostatinantibodies.

It is an object of present invention to provide a method for detectingand quantifying the presence of an antibody specific for an angiostatinin a body fluid.

Still another object of the present invention is to provide acomposition consisting of antibodies to angiostatin that are selectivefor specific regions of the angiostatin molecule that do not recognizeplasminogen.

It is another object of the present invention to provide a method forthe detection or prognosis of cancer.

It is another object of the present invention to provide a compositionfor use in visualizing and quantitating sites of angiostatin binding invivo and in vitro.

It is yet another object of the present invention to provide acomposition for use in detection and quantification of angiostatinbiosynthesis.

It is yet another object of the present invention to provide a therapyfor cancer that has minimal side effects.

Still another object of the present invention is to provide acomposition comprising angiostatin or an angiostatin peptide linked to acytotoxic agent for treating or repressing the growth of a cancer.

Another object of the present invention is to provide a method fortargeted delivery of angiostatin-related compositions to specificlocations.

Yet another object of the invention is to provide compositions andmethods useful for gene therapy for the modulation of angiogenicprocesses.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B shows SEQ ID NO:1, the amino acid sequence of the wholemurine plasminogen.

FIGS. 2A, 2B and 2C show the beginning sequence of the angiostatin formurine (SEQ ID NO:2) and compares the murine sequence with correspondinghuman (SEQ ID NO:3), Rhesus monkey (SEQ ID NO:4), porcine (SEQ ID NO:5)and bovine (SEQ ID NO:6) plasminogen peptide fragments. The mousesequence is listed first, followed by human, Rhesus, porcine and bovine.

FIG. 3A shows BrdU labeling index of tumor cells in the lung in thepresence or absence of a primary tumor. FIG. 3B shows mouse lung weightsin the presence or absence of a primary tumor.

FIG. 4 shows Matrigel analysis of the influence of a Lewis lung primarytumor on bFGF driven angiogenesis in vivo.

FIG. 5 shows dose response curve for serum derived from mice bearingLewis lung carcinoma (LLC-Low) versus serum from normal mice. Bovinecapillary endothelial cells were assayed in a bFGF-driven 72-hourproliferation assay.

FIGS. 6A and 6B show that both low (FIG. 6A) and high (FIG. 6B)metastatic tumors contain endothelial mitogenic activity in theirascites, but only the low metastatic tumor line has endothelialinhibitory activity in the serum.

FIGS. 7A and 7B show a Reserve Phase Chromatograpic profice of partiallypurified serum (FIG. 7A) or urine (FIG. 7B) from tumor-bearing animals.

FIG. 8 shows surface lung metastases after the 13 day treatment of micewith intact plasminogen molecule, active fraction from a lysine bindingsite I preparation of human plasminogen, concentrated urine from tumorbearing mice and concentrated urine from normal mice.

FIG. 9 shows lung weight after the 13 day treatment of mice with intactplasminogen molecule of human plasminogen, active fraction from lysinebinding site I preparation, concentrated urine from tumor bearing miceand concentrated urine from normal mice.

FIG. 10 is a schematic representation of the pTrcHis vector.

FIG. 11 depicts an immunoblot of E. coli expressed human angiostatinfrom a 10 L scaled-up fermentation, probed with monoclonal antibodyagainst human plasminogen kringle region 1-3. Arrow shows recombinanthuman angiostatin. A) shows recombinant angiostatin eluted with 0.2 Mamino caproic acid; B) shows the last wash with 1 X PBS of the lysinecolumn; and C) shows clarified lysate from cracked cells.

FIG. 12. Is a graph depicting percent inhibition of growing bovinecapillary endothelial cells as a function of dilution of stock; A1, A2,B1, B2, and E are recombinant clones that express human angiostatinanti-angiogenesis activity; C1, C2, D1 and D2 controls are negativecontrols clones containing vector only without the human DNA sequencecoding for angiostatin.

FIG. 13 shows the inhibitory effect on proliferation of recombinanthuman angiostatin on bovine capillary endothelial cells in vitro.

FIG. 14 shows the effect of removal of Lewis lung carcinoma primarytumor on the growth of its lung metastases. Panels 14a, 14b and 14c showgrowth of lung metastases after removal of a primary tumor. Panels 14d,14e and 14f show suppression of metastatic growth when the primary tumoris left intact. Animals were killed 5 days (panels 14a and 14d), 10 days(panels 14b and 14e) and 15 days (panels 14c and 14f) after operation.Note the presence of micrometastases (arrows) in the lungs of mice shownin panels 14d, 14e and 14f. Scale bar, 1 mm.

FIGS. 15A, 15B and 15C show an analysis of the lung weight (panel 15a),BrdU labeling index (panel 15b) and apoptotic index (panel 15c) ofmetastases in the lungs of mice with the primary Lewis lung tumor intact(solid bars) or removed (blank bars).

FIGS. 16A, 16B and 16C show an analysis of the lung weight (panel 16a),BrdU labeling index (panel 16b) and apoptotic index (panel 16c) ofmetastases in the lungs of mice with removed primary Lewis lung tumorstreated with TNP-1470 (solid bars) or saline (blank bars).

FIG. 17 shows the inhibition of growth of a T241 primary tumor in miceby treatment with human angiostatin in vivo with a single injection of40 mg/kg/day.

FIG. 18 shows the inhibition of growth of a LLC-LM primary tumor in miceby treatment with human angiostatin in vivo at two doses of 40mg/kg/dose (80 mg/kg/day).

FIG. 19 shows the effect of administration of angiostatin protein tomice having implated T241 fibrosarcoma cells on total tumor volume as afunction of time.

FIG. 20 shows the effect of administration of angiostatin protein tomice having implated Lewis lung carcinoma (LM) cells on total tumorvolume as a function of time.

FIG. 21 shows the effect of administration of angiostatin protein tomice having implated reticulum cell sarcoma cells on total tumor volumeas a function of time.

FIG. 22 shows the effect of administration of angiostatin protein toimmunodeficient SCID mice having implated human prostate carcinoma PC-3cells on total tumor volume as a function of time over a 24 day period.

FIG. 23 shows the effect of administration of angiostatin protein toimmunodeficient SCID mice having implated human breast carcinoma MDA-MBcells on total tumor volume as a function of time over a 24 day period.

FIG. 24 is a schematic representation of cloning of the mouse DNAsequence coding for mouse angiostatin protein derived from mouseplasminogen cDNA. The mouse angiostatin encompasses mouse plasminogenkringle regions 1-4. PCR means polymerase chain reaction; P1 is the5'-end oligonucleotide primre for PCR; P2 is the 3'-end oligonucleotideprimre for PCR; SS designates the signal sequence; ATG is thetranslation initiation codon; TAA is the translation stop codon; HArepresents the hemagglutinin epitope tag (YPYDVPDYASL); K1, K2, K3 andK4 represent mouse plasminogen kringle regions 1, 2, 3 and 4respectively. CMV is the cytomegalovirus promoter; T7 is the bacteriaphage promoter; PA represents pre-activation peptides; and SP6 is the Sp6 promoter.

FIGS. 25A and 25B depict the number of cells as a function of days fornon-transfected cells (mock); cells transfected with the vector alone,without the DNA sequence coding for angiostatin (Vector 5), and twoangiostatin expressing clones (AST 31 and AST 37). FIG. 25A representsthe results of transfection of T241 cells. FIG. 25B represents theresults of LL2 cells.

FIGS. 26A, 26B and 26C show the results of culture medium derived fromE. coli cells containing the angiostatin clone on cell number.Non-transfected cells (mock); cells transfected with the vector alone,without the DNA sequence coding for angiostatin (Vector 5), and threeangiostatin expressing clones (AST 25, AST 31 and AST 37). FIG. 26Arepresents the results of incubation of culture medium from control(mock) and all angiostatin clones (expressing and non-expressing) oncell number. FIG. 26B represents the results of incubation of culturemedium from control (mock), vector alone (vector 6) and angiostatinclones expressing mouse angiostatin on cell number. FIG. 26C representsthe results of incubation of purified culture medium from control (mock)and angiostatin clones expressing mouse angiostatin on cell number,wherein the culture medium was purified over a lysine-sepharose columeto yield lysine binding components.

FIG. 27 shows the effect on total tumor volume as a function of time ofimplanting T241 fibrosarcoma cells in mice, where the fibrosarcoma cellshave been transfected with a vector containing a DNA sequence coding forangiostatin protein, and where the vector is capable of expressingangiostatin protein. "Non-transfected" represents unaltered T241fibrosarcoma cells implanted in mice. "Vector 6" represents T241fibrosarcoma cells transfected with the vector only, which does notcontain the DNA sequence coding for angiostatin protein, implanted inmice. "Clone 25, Clone 31 and Clone 37" represent threeangiostatin-producing clones of T241 fibrosarcoma cells transfected witha vector containg the DNA sequence coding for angiostation proteinimplanted in mice.

DETAILED DESCRIPTION

The present invention includes compositions and methods for thedetection and treatment of diseases and processes that are mediated byor associated with angiogenesis. The composition is angiostatin, whichcan be isolated from body fluids including, but not limited to, serum,urine and ascites, or synthesized by chemical or biological methods(e.g. cell culture, recombinant gene expression, peptide synthesis, andin vitro enzymatic catalysis of plasminogen or plasmin to yield activeangiostatin). Recombinant techniques include gene amplification from DNAsources using the polymerase chain reaction (PCR), and geneamplification from RNA sources using reverse transcriptase/PCR.Angiostatin inhibits the growth of blood vessels into tissues such asunvascularized or vascularized tumors.

The present invention also encompasses a composition comprising, avector containing a DNA sequence encoding angiostatin, wherein thevector is capable of expressing angiostatin when present in a cell, acomposition comprising a cell containing a vector, wherein the vectorcontains a DNA sequence encoding angiostatin or fragments or analogsthereof, and wherein the vector is capable of expressing angiostatinwhen present in the cell, and a method comprising, implanting into ahuman or non-human animal a cell containing a vector, wherein the vectorcontains a DNA sequence encoding angiostatin, and wherein the vector iscapable of expressing angiostatin when present in the cell.

Still further, the present invention encompasses angiostatin,angiostatin fragments, angiostatin antisera, angiostatin receptoragonists or angiostatin receptor antagonists that are combined withpharmaceutically acceptable excipients, and optionally sustained-releasecompounds or compositions, such as biodegradable polymers, to formtherapeutic compositions. In particular, the invention includes acomposition comprising an antibody that specifically binds toangiostatin, wherein the antibody does not bind to plasminogen.

More particularly, the present invention includes a protein designatedangiostatin that has a molecular weight of approximately 38 to 45kilodaltons (kD) that is capable of overcoming the angiogenic activityof endogenous growth factors such as bFGF, in vitro. Angiostatin is aprotein having a molecular weight of between approximately 38kilodaltons and 45 kilodaltons as determined by reducing polyacrylamidegel electrophoresis and having an amino acid sequence substantiallysimilar to that of a murine plasminogen fragment beginning at amino acidnumber 98 of an intact murine plasminogen molecule. The term"substantially similar," when used in reference to angiostatin aminoacid sequences, means an amino acid sequence having anti-angiogenicactivity and having a molecular weight of approximately 38 kD to 45 kD,which also has a high degree of sequence homology to the peptidefragment of mouse plasminogen beginning approximately at amino acidnumber 98 in mouse plasminogen and weighing 38 kD to 45 kD. A highdegree of homology means at least approximately 60% amino acid homology,desirably at least approximately 70% amino acid homology, and moredesirably at least approximately 80% amino acid homology. The term"endothelial inhibiting activity" as used herein means the capability ofa molecule to inhibit angiogenesis in general and, for example, toinhibit the growth of bovine capillary endothelial cells in culture inthe presence of fibroblast growth factor.

The amino acid sequence of the complete murine plasminogen molecule isshown in FIGS. 1A and 1B and in SEQ ID NO:1, The sequence forangiostatin begins approximately at amino acid 98. Active humanangiostatin may start at either amino acid 97 or 99 of the intact humanplasminogen molecule. The amino acid sequence of the first 339 aminoacids of angiostatin from mouse is shown in FIG. 2A, 2B and 2C (SEQ IDNO:2), and is compared with the sequences of corresponding plasminogenpeptide fragments from human (SEQ ID NO:3, Rhesus monkey (SEQ ID NO:4),porcine (SEQ ID NO:5) and bovine (SEQ ID NO:6) plasminogen. Given thatthese sequences are identical in well over 50% of their amino acids, itis to be understood that the amino acid sequence of the angiostatin issubstantially similar among species. The total number of amino acids inangiostatin is not known precisely but is defined by the molecularweight of the active molecule. The amino acid sequence of theangiostatin of the present invention may vary depending upon from whichspecies the plasminogen molecule is derived. Thus, although theangiostatin of the present invention that is derived from humanplasminogen has a slightly different sequence than angiostatin derivedfrom mouse, it has anti-angiogenic activity as shown in a mouse tumormodel.

Angiostatin has been shown to be capable of inhibiting the growth ofendothelial cells in vitro. Angiostatin does not inhibit the growth ofcell lines derived from other cell types. Specifically, angiostatin hasno effect on Lewis lung carcinoma cell lines, mink lung epithelium, 3T3fibroblasts, bovine aortic smooth muscle cells, bovine retinal pigmentepithelium, MDCk cells (canine renal epithelium), WI38 cells (humanfetal lung fibroblasts) EFN cells (murine fetal fibroblasts) and LMcells (murine connective tissue). Endogenous angiostatin in a tumorbering mouse is effective at inhibiting metastases at a systemicconcentration of approximately 10 mg angiostatin/kg body weight.

Angiostatin has a specific three dimensional conformation that isdefined by the kringle region of the plasminogen molecule. (Robbins, K.C., "The plasminogen-plasmin enzyme system" Hemostasis and Thrombosis,Basic Principles and Practice, 2nd Edition, ed. by Colman, R. W. et al.J. B. Lippincott Company, pp. 340-357, 1987) There are five such kringleregions, which are conformationally related motifs and have substantialsequence homology, in the NH₂ terminal portion of the plasminogenmolecule. The three dimensional conformation of angiostatin is believedto encompass plasminogen kringle regions 1 through 3 and a part ofkringle region 4. Each kringle region of the plasminogen moleculecontains approximately 80 amino acids and contains 3 disulfide bonds.This cysteine motif is known to exist in other biologically activeproteins. These proteins include, but are not limited to, prothrombin,hepatocyte growth factor, scatter factor and macrophage stimulatingprotein. (Yoshimura, T, et al., "Cloning, sequencing, and expression ofhuman macrophage stimulating protein (MSP, MST1) confirms MSP as amember of the family of kringle proteins and locates the MSP gene onChromosome 3" J. Biol. Chem., Vol. 268, No. 21, pp. 15461-15468, 1993).It is contemplated that any isolated protein or peptide having a threedimensional kringle-like conformation or cysteine motif that hasanti-angiogenic activity in vivo, is part of the present invention.

The present invention also includes the detection of the angiostatin inbody fluids and tissues for the purpose of diagnosis or prognosis ofdiseases such as cancer. The present invention also includes thedetection of angiostatin binding sites and receptors in cells andtissues. The present invention also includes methods of treating orpreventing angiogenic diseases and processes including, but not limitedto, arthritis and tumors by stimulating the production of angiostatin,and/or by administering substantially purified angiostatin, orangiostatin agonists or antagonists, and/or angiostatin antisera orantisera directed against angiostatin antisera to a patient. Additionaltreatment methods include administration of angiostatin, angiostatinfragments, angiostatin analogs, angiostatin antisera, or angiostatinreceptor agonists and antagonists linked to cytotoxic agents. It is tobe understood that the angiostatin can be animal or human in origin.Angiostatin can also be produced synthetically by chemical reaction orby recombinant techniques in conjunction with expression systems.Angiostatin can also be produced by enzymatically cleaving isolatedplasminogen or plasmin to generate peptides having anti-angiogenicactivity. Angiostatin may also be produced by compounds that mimic theaction of endogenous enzymes that cleave plasminogen to angiostatin.Angiostatin production may also be modulated by compounds that affectthe activity of plasminogen cleaving enxymes.

Passive antibody therapy using antibodies that specifically bindangiostatin can be employed to modulate angiogenic-dependent processessuch as reproduction, development, and wound healing and tissue repair.In addition, antisera directed to the Fab regions of angiostatinantibodies can be administered to block the ability of endogenousangiostatin antisera to bind angiostatin.

The present invention also encompasses gene therapy whereby the geneencoding angiostatin is regulated in a patient. Various methods oftransferring or delivering DNA to cells for expression of the geneproduct protein, otherwise referred to as gene therapy, are disclosed inGene Transfer into Mammalian Somatic Cells in vivo, N. Yang, Crit. Rev.Biotechn. 12(4): 335-356 (1992), which is hereby incorporated byreference. Gene therapy encompasses incorporation of DNA sequences intosomatic cells or germ line cells for use in either ex vivo or in vivotherapy. Gene therapy functions to replace genes, augment normal orabnormal gene function, and to combat infectious diseases and otherpathologies.

Strategies for treating these medical problems with gene therapy includetherapeutic strategies such as identifying the defective gene and thenadding a functional gene to either replace the function of the defectivegene or to augment a slightly functional gene; or prophylacticstrategies, such as adding a gene for the product protein that willtreat the condition or that will make the tissue or organ moresusceptible to a treatment regimen. As an example of a prophylacticstrategy, a gene such as angiostatin may be placed in a patient and thusprevent occurrence of angiogenesis; or a gene that makes tumor cellsmore susceptible to radiation could be inserted and then radiation ofthe tumor would cause increased killing of the tumor cells.

Many protocols for transfer of angiostatin DNA or angiostatin regulatorysequences are envisioned in this invention. Transfection of promotersequences, other than one normally found specifically associated withangiostatin, or other sequences which would increase production ofangiostatin protein are also envisioned as methods of gene therapy. Anexample of this technology is found in Transkaryotic Therapies, Inc., ofCambridge, Mass., using homologous recombination to insert a "geneticswitch" that turns on an erythropoietin gene in cells. See GeneticEngineering News, Apr. 15, 1994. Such "genetic switches" could be usedto activate angiostatin (or the angiostatin receptor) in cells notnormally expressing angiostatin (or the angiostatin receptor).

Gene transfer methods for gene therapy fall into three broadcategories-physical (e.g., electroporation, direct gene transfer andparticle bombardment), chemical (lipid-based carriers, or othernon-viral vectors) and biological (virus-derived vector and receptoruptake). For example, non-viral vectors may be used which includeliposomes coated with DNA. Such liposome/DNA complexes may be directlyinjected intravenously into the patient. It is believed that theliposome/DNA complexes are concentrated in the liver where they deliverthe DNA to macrophages and Kupffer cells. These cells are long lived andthus provide long term expression of the delivered DNA. Additionally,vectors or the "naked" DNA of the gene may be directly injected into thedesired organ, tissue or tumor for targeted delivery of the therapeuticDNA.

Gene therapy methodologies can also be described by delivery site.Fundamental ways to deliver genes include ex vivo gene transfer, in vivogene transfer, and in vitro gene transfer. In ex vivo gene transfer,cells are taken from the patient and grown in cell culture. The DNA istransfected into the cells, the transfected cells are expanded in numberand then reimplanted in the patient. In in vitro gene transfer, thetransformed cells are cells growing in culture, such as tissue culturecells, and not particular cells from a particular patient. These"laboratory cells" are transfected, the transfected cells are selectedand expanded for either implantation into a patient or for other uses.

In vivo gene transfer involves introducing the DNA into the cells of thepatient when the cells are within the patient. Methods include usingvirally mediated gene transfer using a noninfectious virus to deliverthe gene in the patient or injecting naked DNA into a site in thepatient and the DNA is taken up by a percentage of cells in which thegene product protein is expressed. Additionally, the other methodsdescribed herein, such as use of a "gene gun," may be used for in vitroinsertion of angiostatin DNA or angiostatin regulatory sequences.

Chemical methods of gene therapy may involve a lipid based compound, notnecessarily a liposome, to ferry the DNA across the cell membrane.Lipofectins or cytofectins, lipid-based positive ions that bind tonegatively charged DNA, make a complex that can cross the cell membraneand provide the DNA into the interior of the cell. Another chemicalmethod uses receptor-based endocytosis, which involves binding aspecific ligand to a cell surface receptor and enveloping andtransporting it across the cell membrane. The ligand binds to the DNAand the whole complex is transported into the cell. The ligand genecomplex is injected into the blood stream and then target cells thathave the receptor will specifically bind the ligand and transport theligand-DNA complex into the cell.

Many gene therapy methodologies employ viral vectors to insert genesinto cells. For example, altered retrovirus vectors have been used in exvivo methods to introduce genes into peripheral and tumor-infiltratinglymphocytes, hepatocytes, epidermal cells, myocytes, or other somaticcells. These altered cells are then introduced into the patient toprovide the gene product from the inserted DNA.

Viral vectors have also been used to insert genes into cells using invivo protocols. To direct tissue-specific expression of foreign genes,cis-acting regulatory elements or promoters that are known to be tissuespecific can be used. Alternatively, this can be achieved using in situdelivery of DNA or viral vectors to specific anatomical sites in vivo.For example, gene transfer to blood vessels in vivo was achieved byimplanting in vitro transduced endothelial cells in chosen sites onarterial walls. The virus infected surrounding cells which alsoexpressed the gene product. A viral vector can be delivered directly tothe in vivo site, by a catheter for example, thus allowing only certainareas to be infected by the virus, and providing long-term, sitespecific gene expression. In vivo gene transfer using retrovirus vectorshas also been demonstrated in mammary tissue and hepatic tissue byinjection of the altered virus into blood vessels leading to the organs.

Viral vectors that have been used for gene therapy protocols include butare not limited to, retroviruses, other RNA viruses such as poliovirusor Sindbis virus, adenovirus, adeno-associated virus, herpes viruses, SV40, vaccinia and other DNA viruses. Replication-defective murineretroviral vectors are the most widely utilized gene transfer vectors.Murine leukemia retroviruses are composed of a single strand RNAcomplexed with a nuclear core protein and polymerase (pol) enzymes,encased by a protein core (gag) and surrounded by a glycoproteinenvelope (env) that determines host range. The genomic structure ofretroviruses include the gag, pol, and env genes enclosed at by the 5'and 3' long terminal repeats (LTR). Retroviral vector systems exploitthe fact that a minimal vector containing the 5' and 3' LTRs and thepackaging signal are sufficient to allow vector packaging, infection andintegration into target cells providing that the viral structuralproteins are supplied in trans in the packaging cell line. Fundamentaladvantages of retroviral vectors for gene transfer include efficientinfection and gene expression in most cell types, precise single copyvector integration into target cell chromosomal DNA, and ease ofmanipulation of the retroviral genome.

The adenovirus is composed of linear, double stranded DNA complexed withcore proteins and surrounded with capsid proteins. Advances in molecularvirology have led to the ability to exploit the biology of theseorganisms to create vectors capable of transducing novel geneticsequences into target cells in vivo. Adenoviral-based vectors willexpress gene product peptides at high levels. Adenoviral vectors havehigh efficiencies of infectivity, even with low titers of virus.Additionally, the virus is fully infective as a cell free virion soinjection of producer cell lines are not necessary. Another potentialadvantage to adenoviral vectors is the ability to achieve long termexpression of heterologous genes in vivo.

Mechanical methods of DNA delivery include fusogenic lipid vesicles suchas liposomes or other vesicles for membrane fusion, lipid particles ofDNA incorporating cationic lipid such as lipofectin, polylysine-mediatedtransfer of DNA, direct injection of DNA, such as microinjection of DNAinto germ or somatic cells, pneumatically delivered DNA-coatedparticles, such as the gold particles used in a "gene gun," andinorganic chemical approaches such as calcium phosphate transfection.Another method, ligand-mediated gene therapy, involves complexing theDNA with specific ligands to form ligand-DNA conjugates, to direct theDNA to a specific cell or tissue.

It has been found that injecting plasmid DNA into muscle cells yieldshigh percentage of the cells which are transfected and have sustainedexpression of marker genes. The DNA of the plasmid may or may notintegrate into the genome of the cells. Non-integration of thetransfected DNA would allow the transfection and expression of geneproduct proteins in terminally differentiated, non-proliferative tissuesfor a prolonged period of time without fear of mutational insertions,deletions, or alterations in the cellular or mitochondrial genome.Long-term, but not necessarily permanent, transfer of therapeutic genesinto specific cells may provide treatments for genetic diseases or forprophylactic use. The DNA could be reinjected periodically to maintainthe gene product level without mutations occurring in the genomes of therecipient cells. Non-integration of exogenous DNAs may allow for thepresence of several different exogenous DNA constructs within one cellwith all of the constructs expressing various gene products.

Particle-mediated gene transfer methods were first used in transformingplant tissue. With a particle bombardment device, or "gene gun," amotive force is generated to accelerate DNA-coated high densityparticles (such as gold or tungsten) to a high velocity that allowspenetration of the target organs, tissues or cells. Particle bombardmentcan be used in in vitro systems, or with ex vivo or in vivo techniquesto introduce DNA into cells, tissues or organs.

Electroporation for gene transfer uses an electrical current to makecells or tissues susceptible to electroporation-mediated gene transfer.A brief electric impulse with a given field strength is used to increasethe permeability of a membrane in such a way that DNA molecules canpenetrate into the cells. This technique can be used in in vitrosystems, or with ex vivo or in vivo techniques to introduce DNA intocells, tissues or organs.

Carrier mediated gene transfer in vivo can be used to transfect foreignDNA into cells. The carrier-DNA complex can be conveniently introducedinto body fluids or the bloodstream and then site specifically directedto the target organ or tissue in the body. Both liposomes andpolycations, such as polylysine, lipofectins or cytofectins, can beused. Liposomes can be developed which are cell specific or organspecific and thus the foreign DNA carried by the liposome will be takenup by target cells. Injection of immunoliposomes that are targeted to aspecific receptor on certain cells can be used as a convenient method ofinserting the DNA into the cells bearing the receptor. Another carriersystem that has been used is the asialoglycoportein/polylysine conjugatesystem for carrying DNA to hepatocytes for in vivo gene transfer.

The transfected DNA may also be complexed with other kinds of carriersso that the DNA is carried to the recipient cell and then resides in thecytoplasm or in the nucleoplasm. DNA can be coupled to carrier nuclearproteins in specifically engineered vesicle complexes and carrieddirectly into the nucleus.

Gene regulation of angiostatin may be accomplished by administeringcompounds that bind to the angiostatin gene, or control regionsassociated with the angiostatin gene, or its corresponding RNAtranscript to modify the rate of transcription or translation.Additionally, cells transfected with a DNA sequence encoding angiostatinmay be administered to a patient to provide an in vivo source ofangiostatin. For example, cells may be transfected with a vectorcontaining a nucleic acid sequence encoding angiostatin.

The term "vector" as used herein means a carrier that can contain orassociate with specific nucleic acid sequences, which functions totransport the specific nucleic acid sequences into a cell. Examples ofvectors include plasmids and infective microorganisms such as viruses,or non-viral vectors such as ligand-DNA conjugates, liposomes, lipid-DNAcomplexes. It may be desirable that a recombinant DNA moleculecomprising an angiostatin DNA sequence is operatively linked to anexpression control sequence to form an expression vector capable ofexpressing angiostatin. The transfected cells may be cells derived fromthe patient's normal tissue, the patient's diseased tissue, or may benon-patient cells.

For example, tumor cells removed from a patient can be transfected witha vector capable of expressing the angiostatin protein of the presentinvention, and re-introduced into the patient. The transfected tumorcells produce angiostatin levels in the patient that inhibit the growthof the tumor. Patients may be human or non-human animals. Cells may alsobe transfected by non-vector, or physical or chemical methods known inthe art such as electroporation, ionoporation, or via a "gene gun."Additionally, angiostatin DNA may be directly injected, without the aidof a carrier, into a patient. In particular, angiostatin DNA may beinjected into skin, muscle or blood.

The gene therapy protocol for transfecting angiostatin into a patientmay either be through integration of the angiostatin DNA into the genomeof the cells, into minichromosomes or as a separate replicating ornon-replicating DNA construct in the cytoplasm or nucleoplasm of thecell. Angiostatin expression may continue for a long-period of time ormay be reinjected periodically to maintain a desired level of theangiostatin protein in the cell, the tissue or organ or a determinedblood level.

Angiostatin can be isolated on an HPLC C4 column (see Table 3). Theangiostatin protein is eluted at 30 to 35% in an acetonitrile gradient.On a sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE)gel under reducing conditions, the protein band with activity eluted asa single peak at approximately 38 kilodaltons.

The inventors have shown that a growing primary tumor is associated withthe release into the blood stream of specific inhibitor(s) ofendothelial cell proliferation, including angiostatin which can suppressangiogenesis within a metastasis and thereby inhibit the growth of themetastasis itself. The source of the angiostatin associated with theprimary tumor is not known. The compound may be produced by degradationof plasminogen by a specific protease, or angiostatin could be producedby expression of a specific gene coding for angiostatin.

The angiogenic phenotype of a primary tumor depends on production ofangiogenic peptides in excess of endothelial cell inhibitors which areelaborated by normal cells, but are believed to be down-regulated duringtransformation to neoplasia. While production of angiostatin may bedown-regulated in an individual tumor cell relative to production by itsparent cell type, the total amount of inhibitor elaborated by the wholetumor may be sufficient to enter the circulation and suppressendothelial growth at remote sites of micrometastases. Angiostatinremains in the circulation for a significantly longer time than theangiogenic peptide(s) released by a primary tumor. Thus, the angiogenicpeptides appear to act locally, whereas angiostatin acts globally andcirculates in the blood with a relatively long half-life. The half-lifeof the angiostatin is approximately 12 hours to 5 days.

Although not wanting to be bound by the following hypothesis, it isbelieved that when a tumor becomes angiogenic it releases one or moreangiogenic peptides (e.g., aFGF, bFGF, VEGF, IL-8, GM-CSF, etc.), whichact locally, target endothelium in the neighborhood of a primary tumorfrom an extravascular direction, and do not circulate (or circulate witha short half-life). These angiogenic peptides must be produced in anamount sufficient to overcome the action of endothelial cell inhibitor(inhibitors of angiogenesis) for a primary tumor to continue to expandits population. Once such a primary tumor is growing well, it continuesto release endothelial cell inhibitors into the circulation. Accordingto this hypothesis, these inhibitors act remotely at a distance from theprimary tumor, target capillary endothelium of a metastasis from anintravascular direction, and continue to circulate. Thus, just at thetime when a remote metastasis might begin to initiate angiogenesis, thecapillary endothelium in its neighborhood could be inhibited by incomingangiostatin.

Once a primary tumor has reached sufficient size to cause angiostatin tobe released continuously into the circulation, it is difficult for asecond tumor implant (or a micrometastasis) to initiate or increase itsown angiogenesis. If a second tumor implant (e.g., into the subcutaneousspace, or into the cornea, or intravenously to the lung) occurs shortlyafter the primary tumor is implanted, the primary tumor will not be ableto suppress the secondary tumor (because angiogenesis in the secondarytumor will already be well underway). If two tumors are implantedsimultaneously (e.g., in opposite flanks), the inhibitors may have anequivalent inhibiting effect on each other.

The angiostatin of the present invention can be:

(i) Administered to tumor-bearing humans or animals as anti-angiogenictherapy;

(ii) Monitored in human or animal serum, urine, or tissues as prognosticmarkers; and

(iii) Used as the basis to analyze serum and urine of cancer patientsfor similar angiostatic molecules.

It is contemplated as part of the present invention that angiostatin canbe isolated from a body fluid such as blood or urine of patients or theangiostatin can be produced by recombinant DNA methods or syntheticpeptide chemical methods that are well known to those of ordinary skillin the art. Protein purification methods are well known in the art and aspecific example of a method for purifying angiostatin, and assaying forinhibitor activity is provided in the examples below. Isolation of humanendogenous angiostatin is accomplished using similar techniques.

One example of a method of producing angiostatin using recombinant DNAtechniques entails the steps of (1) identifying and purifyingangiostatin as discussed above, and as more fully described below, (2)determining the N-terminal amino acid sequence of the purifiedinhibitor, (3) synthetically generating 5' and 3' DNA oligonucleotideprimers for the angiostatin sequence, (4) amplifying the angiostatingene sequence using polymerase, (5) inserting the amplified sequenceinto an appropriate vector such as an expression vector, (6) insertingthe gene containing vector into a microorganism or other expressionsystem capable of expressing the inhibitor gene, and (7) isolating therecombinantly produced inhibitor. Appropriate vectors include viral,bacterial and eukaryotic (such as yeast) expression vectors. The abovetechniques are more fully described in laboratory manuals such as"Molecular Cloning: A Laboratory Manual" Second Edition by Sambrook etal., Cold Spring Harbor Press, 1989. The DNA sequence of humanplasminogen has been published (Browne, M. J., et al., "Expression ofrecombinant human plasminogen and aglycoplasminogen in HeLa cells"Fibrinolysis Vol.5 (4). 257-260, 1991) and is incorporated herein byreference. One example of such a recombinant expression system isEscherichia coli strain XL-1B pTrcHisA/HAsH4 available upon permissionfrom the American Type Culture Collection 12301 Parklawn Drive,Rockville, Md. 20852 under ATCC Designation No. 98231 deposited on Oct.23, 1996.

The gene for angiostatin may also be isolated from cells or tissue (suchas tumor cells) that express high levels of angiostatin by (1) isolatingmessenger RNA from the tissue, (2) using reverse transcriptase togenerate the corresponding DNA sequence and then (3) using thepolymerase chain reaction (PCR) with the appropriate primers to amplifythe DNA sequence coding for the active angiostatin amino acid sequence.

Yet another method of producing angiostatin, or biologically activefragments thereof, is by peptide synthesis. Once a biologically activefragment of an angiostatin is found using the assay system describedmore fully below, it can be sequenced, for example by automated peptidesequencing methods. Alternatively, once the gene or DNA sequence whichcodes for angiostatin is isolated, for example by the methods describedabove, the DNA sequence can be determined using manual or automatedsequencing methods well know in the art. The nucleic acid sequence inturn provides information regarding the amino acid sequence. Thus, ifthe biologically active fragment is generated by specific methods, suchas tryptic digests, or if the fragment is N-terminal sequenced, theremaining amino acid sequence can be determined from the correspondingDNA sequence.

Once the amino acid sequence of the peptide is known, the fragment canbe synthesized by techniques well known in the art, as exemplified by"Solid Phase Peptide Synthesis: A Practical Approach" E. Atherton and R.C. Sheppard, IRL Press, Oxford, England. Similarly, multiple fragmentscan be synthesized which are subsequently linked together to form largerfragments. These synthetic peptide fragments can also be made with aminoacid substitutions at specific locations to test for agonistic andantagonistic activity in vitro and in vivo. Peptide fragments thatpossess high affinity binding to tissues can be used to isolate theangiostatin receptor on affinity columns. Isolation and purification ofthe angiostatin receptor is a fundamental step towards elucidating themechanism of action of angiostatin. Isolation of an angiostatin receptorand identification of angiostatin agonists and antagonists willfacilitate development of drugs to modulate the activity of theangiostatin receptor, the final pathway to biological activity.Isolation of the receptor enables the construction of nucleotide probesto monitor the location and synthesis of the receptor, using in situ andsolution hybridization technology. Further, the gene for the angiostatinreceptor can be isolated, incorporated into an expression vector andtransfected into cells, such as patient tumor cells to increase theability of a cell type, tissue or tumor to bind angiostatin and inhibitlocal angiogenesis.

Angiostatin is effective in treating diseases or processes that aremediated by, or involve, angiogenesis. The present invention includesthe method of treating an angiogenesis mediated disease with aneffective amount of angiostatin, or a biologically active fragmentthereof, or combinations of angiostatin fragmetns that collectivelypossess anti-angiogenic activity, or angiostatin agonists andantagonists. The angiogenesis mediated diseases include, but are notlimited to, solid tumors; blood born tumors such as leukemias; tumormetastasis; benign tumors, for example hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis;psoriasis; ocular angiogenic diseases, for example, diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;and wound granulation. Angiostatin is useful in the treatment of diseaseof excessive or abnormal stimulation of endothelial cells. Thesediseases include, but are not limited to, intestinal adhesions, Crohn'sdisease, atherosclerosis, scleroderma, and hypertrophic scars, i.e.,keloids. Angiostatin can be used as a birth control agent by preventingvascularization required for embryo implantation. Angiostatin is usefulin the treatment of diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa) andulcers (Helicobacter pylon).

The synthetic peptide fragments of angiostatin have a variety of uses.The peptide that binds to the angiostatin receptor with high specificityand avidity is radiolabeled and employed for visualization andquantitation of binding sites using autoradiographic and membranebinding techniques. This application provides important diagnostic andresearch tools. Knowledge of the binding properties of the angiostatinreceptor facilitates investigation of the transduction mechanisms linkedto the receptor.

In addition, labeling angiostatin peptides with short lived isotopesenables visualization of receptor binding sites in vivo using positronemission tomography or other modern radiographic techniques to locatetumors with angiostatin binding sites.

Systematic substitution of amino acids within these synthesized peptidesyields high affinity peptide agonists and antagonists to the angiostatinreceptor that enhance or diminish angiostatin binding to its receptor.Such agonists are used to suppress the growth of micrometastases,thereby limiting the spread of cancer. Antagonists to angiostatin areapplied in situations of inadequate vascularization, to block theinhibitory effects of angiostatin and promote angiogenesis. For example,this treatment may have therapeutic effects to promote wound healing indiabetics.

Angiostatin peptides are employed to develop affinity columns forisolation of the angiostatin receptor from cultured tumor cells.Isolation and purification of the angiostatin receptor is followed byamino acid sequencing. Using this information the gene or genes codingfor the angiostatin receptor can be identified and isolated. Next,cloned nucleic acid sequences are developed for insertion into vectorscapable of expressing the receptor. These techniques are well known tothose skilled in the art. Transfection of the nucleic acid sequence(s)coding for angiostatin receptor into tumor cells, and expression of thereceptor by the transfected tumor cells enhances the responsiveness ofthese cells to endogenous or exogenous angiostatin and therebydecreasing the rate of metastatic growth.

Cytotoxic agents such as ricin, are linked to angiostatin, and highaffinity angiostatin peptide fragments, thereby providing a tool fordestruction of cells that bind angiostatin. These cells may be found inmany locations, including but not limited to, micrometastases andprimary tumors. Peptides linked to cytotoxic agents are infused in amanner designed to maximize delivery to the desired location. Forexample, ricin-linked high affinity angiostatin fragments are deliveredthrough a cannula into vessels supplying the target site or directlyinto the target. Such agents are also delivered in a controlled mannerthrough osmotic pumps coupled to infusion cannulae. A combination ofangiostatin antagonists may be co-applied with stimulators ofangiogenesis to increase vascularization of tissue. This therapeuticregimen provides an effective means of destroying metastatic cancer.

Angiostatin may be used in combination with other compositions andprocedures for the treatment of diseases. For example, a tumor may betreated conventionally with surgery, radiation or chemotherapy combinedwith angiostatin and then angiostatin may be subsequently administeredto the patient to extend the dormancy of micrometastases and tostabilize and inhibit the growth of any residual primary tumor.Additionally, angiostatin, angiostatin fragments, angiostatin antisera,angiostatin receptor agonists, angiostatin receptor antagonists, orcombinations thereof, are combined with pharmaceutically acceptableexcipients, and optionally sustained-release matrix, such asbiodegradable polymers, to form therapeutic compositions.

A sustained-release matrix, as used herein, is a matrix made ofmaterials, usually polymers, which are degradable by enzymatic oracid/base hydrolysis or by dissolution. Once inserted into the body, thematrix is acted upon by enzymes and body fluids. The sustained-releasematrix desirably is chosen from biocompatible materials such asliposomes, polylactides (polylactic acid), polyglycolide (polymer ofglycolic acid), polylactide co-glycolide (co-polymers of lactic acid andglycolic acid) polyanhydrides, poly(ortho)esters, polypeptides,hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fattyacids, phospholipids, polysaccharides, nucleic acids, polyamino acids,amino acids such as phenylalanine, tyrosine, isoleucine,polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.A preferred biodegradable matrix is a matrix of one of eitherpolylactide, polyglycolide, or polylactide co-glycolide (co-polymers oflactic acid and glycolic acid).

The angiogenesis-modulating therapeutic composition of the presentinvention may be a solid, liquid or aerosol and may be administered byany known route of administration. Examples of solid therapeuticcompositions include pills, creams, and implantable dosage units. Thepills may be administered orally, the therapeutic creams may beadministered topically. The implantable dosage unitst may beadministered locally, for example at a tumor site, or which may beimplanted for systemic release of the therapeuticangiogenesis-modulating composition, for example subcutaneously.Examples of liquid composition include formulations adapted forinjection subcutaneously, intravenously, intraarterially, andformulations for topical and intraocular administration. Examples ofaersol formulation include inhaler formulation for administration to thelungs.

The angiostatin of the present invention also can be used to generateantibodies that are specific for the inhibitor and its receptor. Theantibodies can be either polyclonal antibodies or monoclonal antibodies.These antibodies that specifically bind to the angiostatin orangiostatin receptors can be used in diagnostic methods and kits thatare well known to those of ordinary skill in the art to detect orquantify the angiostatin or angiostatin receptors in a body fluid ortissue. Results from these tests can be used to diagnose or predict theoccurrence or recurrence of a cancer and other angiogenic mediateddiseases.

The angiostatin also can be used in a diagnostic method and kit todetect and quantify antibodies capable of binding angiostatin. Thesekits would permit detection of circulating angiostatin antibodies whichindicates the spread of micrometastases in the presence of angiostatinsecreted by primary tumors in situ. Patients that have such circulatinganti-angiostatin antibodies may be more likely to develop multipletumors and cancers, and may be more likely to have recurrences of cancerafter treatments or periods of remission. The Fab fragments of theseanti-angiostatin antibodies may be used as antigens to generateanti-angiostatin Fab-fragment antisera which can be used to neutralizeanti-angiostatin antibodies. Such a method would reduce the removal ofcirculating angiostatin by anti-angiostatin antibodies, therebyeffectively elevating circulating angiostatin levels.

Another aspect of the present invention is a method of blocking theaction of excess endogenous angiostatin. This can be done by passivelyimmunizing a human or animal with antibodies specific for the undesiredangiostatin in the system. This treatment can be important in treatingabnormal ovulation, menstruation and placentation, and vasculogenesis.This provides a useful tool to examine the effects of angiostatinremoval on metastatic processes. The Fab fragment of angiostatinantibodies contains the binding site for angiostatin. This fragment isisolated from angiostatin antibodies using techniques known to thoseskilled in the art. The Fab fragments of angiostatin antisera are usedas antigens to generate production of anti-Fab fragment serum. Infusionof this antiserum against the Fab fragments of angiostatin preventsangiostatin from binding to angiostatin antibodies. Therapeutic benefitis obtained by neutralizing endogenous anti-angiostatin antibodies byblocking the binding of angiostatin to the Fab fragments ofanti-angiostatin. The net effect of this treatment is to facilitate theability of endogenous circulating angiostatin to reach target cells,thereby decreasing the spread of metastases.

It is to be understood that the present invention is contemplated toinclude any derivatives of the angiostatin that have endothelialinhibitory activity. The present invention includes the entireangiostatin protein, derivatives of the angiostatin protein andbiologically-active fragments of the angiostatin protein. These includeproteins with angiostatin activity that have amino acid substitutions orhave sugars or other molecules attached to amino acid functional groups.The present invention also includes genes that code for angiostatin andthe angiostatin receptor, and to proteins that are expressed by thosegenes.

The proteins and protein fragments with the angiostatin activitydescribed above can be provided as isolated and substantially purifiedproteins and protein fragments in pharmaceutically acceptableformulations using formulation methods known to those of ordinary skillin the art. These formulations can be administered by standard routes.In general, the combinations may be administered by the topical,transdermal, intraperitoneal, intracranial, intracerebroventricular,intracerebral, intravaginal, intrauterine, oral, rectal or parenteral(e.g., intravenous, intraspinal, subcutaneous or intramuscular) route.In addition, the angiostatin may be incorporated into biodegradablepolymers allowing for sustained release of the compound, the polymersbeing implanted in the vicinity of where drug delivery is desired, forexample, at the site of a tumor or implanted so that the angiostatin isslowly released systemically. Osmotic minipumps may also be used toprovide controlled delivery of high concentrations of angiostatinthrough cannulae to the site of interest, such as directly into ametastatic growth or into the vascular supply to that tumor. Thebiodegradable polymers and their use are described, for example, indetail in Brem et al., J. Neurosurg. 74:441-446 (1991), which is herebyincorporated by reference in its entirety.

The dosage of the angiostatin of the present invention will depend onthe disease state or condition being treated and other clinical factorssuch as weight and condition of the human or animal and the route ofadministration of the compound. For treating humans or animals, betweenapproximately 0.5 mg/kilogram to 500 mg/kilogram of the angiostatin canbe administered. Depending upon the half-life of the angiostatin in theparticular animal or human, the angiostatin can be administered betweenseveral times per day to once a week. It is to be understood that thepresent invention has application for both human and veterinary use. Themethods of the present invention contemplate single as well as multipleadministrations, given either simultaneously or over an extended periodof time.

The angiostatin formulations include those suitable for oral, rectal,ophthalmic (including intravitreal or intracameral), nasal, topical(including buccal and sublingual), intrauterine, vaginal or parenteral(including subcutaneous, intraperitoneal, intramuscular, intravenous,intradermal, intracranial, intratracheal, and epidural) administration.The angiostatin formulations may conveniently be presented in unitdosage form and may be prepared by conventional pharmaceuticaltechniques. Such techniques include the step of bringing intoassociation the active ingredient and the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example, water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, or an appropriate fraction thereof, of theadministered ingredient. It should be understood that in addition to theingredients, particularly mentioned above, the formulations of thepresent invention may include other agents conventional in the arthaving regard to the type of formulation in question. Optionally,cytotoxic agents may be incorporated or otherwise combined withangiostatin proteins, or biologically functional peptide fragementsthereof, to provide dual therapy to the patient.

Angiogenesis inhibiting peptides of the present invention can besynthesized in a standard microchemical facility and purity checked withHPLC and mass spectrophotometry. Methods of peptide synthesis, HPLCpurification and mass spectrophotometry are commonly known to thoseskilled in these arts. Angiostatin peptides and angiostatin receptorspeptides are also produced in recombinant E. coli or yeast expressionsystems, and purified with column chromatography.

Different peptide fragments of the intact angiostatin molecule can besynthesized for use in several applications including, but not limitedto the following; as antigens for the development of specific antisera,as agonists and antagonists active at angiostatin binding sites, aspeptides to be linked to, or used in combination with, cytotoxic agentsfor targeted killing of cells that bind angiostatin. The amino acidsequences that comprise these peptides are selected on the basis oftheir position on the exterior regions of the molecule and areaccessible for binding to antisera. The amino and carboxyl termini ofangiostatin, as well as the mid-region of the molecule are representedseparately among the fragments to be synthesized.

These peptide sequences are compared to known sequences using proteinsequence databases such as GenBank, Brookhaven Protein, SWISS-PROT, andPIR to determine potential sequence homologies. This informationfacilitates elimination of sequences that exhibit a high degree ofsequence homology to other molecules, thereby enhancing the potentialfor high specificity in the development of antisera, agonists andantagonists to angiostatin.

Angiostatin and angiostatin derived peptides can be coupled to othermolecules using standard methods. The amino and carboxyl termini ofangiostatin both contain tyrosine and lysine residues and areisotopically and nonisotopically labeled with many techniques, forexample radiolabeling using conventional techniques (tyrosineresidues-chloramine T, iodogen, lactoperoxidase; lysineresidues-Bolton-Hunter reagent). These coupling techniques are wellknown to those skilled in the art. Alternatively, tyrosine or lysine isadded to fragments that do not have these residues to facilitatelabeling of reactive amino and hydroxyl groups on the peptide. Thecoupling technique is chosen on the basis of the functional groupsavailable on the amino acids including, but not limited to amino,sulfhydral, carboxyl, amide, phenol, and imidazole. Various reagentsused to effect these couplings include among others, glutaraldehyde,diazotized benzidine, carbodiimide, and p-benzoquinone.

Angiostatin peptides are chemically coupled to isotopes, enzymes,carrier proteins, cytotoxic agents, fluorescent molecules,chemiluminescent, bioluminescent and other compounds for a variety ofapplications. The efficiency of the coupling reaction is determinedusing different techniques appropriate for the specific reaction. Forexample, radiolabeling of an angiostatin peptide with ¹²⁵ I isaccomplished using chloramine T and Na¹²⁵ I of high specific activity.The reaction is terminated with sodium metabisulfite and the mixture isdesalted on disposable columns. The labeled peptide is eluted from thecolumn and fractions are collected. Aliquots are removed from eachfraction and radioactivity measured in a gamma counter. In this manner,the unreacted Na¹²⁵ I is separated from the labeled angiostatin peptide.The peptide fractions with the highest specific radioactivity are storedfor subsequent use such as analysis of the ability to bind toangiostatin antisera.

Another application of peptide conjugation is for production ofpolyclonal antisera. For example, angiostatin peptides containing lysineresidues are linked to purified bovine serum albumin usingglutaraldehyde. The efficiency of the reaction is determined bymeasuring the incorporation of radiolabeled peptide. Unreactedglutaraldehyde and peptide are separated by dialysis. The conjugate isstored for subsequent use.

Antiserum against angiostatin, angiostatin analogs, peptide fragments ofangiostatin and the angiostatin receptor can be generated. After peptidesynthesis and purification, both monoclonal and polyclonal antisera areraised using established techniques known to those skilled in the art.For example, polyclonal antisera may be raised in rabbits, sheep, goatsor other animals. Angiostatin peptides conjugated to a carrier moleculesuch as bovine serum albumin, or angiostatin itself, is combined with anadjuvant mixture, emulsified and injected subcutaneously at multiplesites on the back, neck, flanks, and sometimes in the footpads. Boosterinjections are made at regular intervals, such as every 2 to 4 weeks.Blood samples are obtained by venipuncture, for example using themarginal ear veins after dilation, approximately 7 to 10 days after eachinjection. The blood samples are allowed to clot overnight at 4 C. andare centrifuged at approximately 2400 X g at 4 C. for about 30 minutes.The serum is removed, aliquoted, and stored at 4 C. for immediate use orat -20 to -90 C. for subsequent analysis.

All serum samples from generation of polyclonal antisera or mediasamples from production of monoclonal antisera are analyzed fordetermination of antibody titer. Titer is established through severalmeans, for example, using dot blots and density analysis, and also withprecipitation of radiolabeled peptide-antibody complexes using proteinA, secondary antisera, cold ethanol or charcoal-dextran followed byactivity measurement with a gamma counter. The highest titer antiseraare also purified on affinity columns which are commercially available.Angiostatin peptides are coupled to the gel in the affinity column.Antiserum samples are passed through the column and anti-angiostatinantibodies remain bound to the column. These antibodies are subsequentlyeluted, collected and evaluated for determination of titer andspecificity.

The highest titer angiostatin antisera is tested to establish thefollowing; a) optimal antiserum dilution for highest specific binding ofthe antigen and lowest non-specific binding, b) the ability to bindincreasing amounts of angiostatin peptide in a standard displacementcurve, c) potential cross-reactivity with related peptides and proteins,including plasminogen and also angiostatin of related species, d)ability to detect angiostatin peptides in extracts of plasma, urine,tissues, and in cell culture media.

Kits for measurement of angiostatin, and the angiostatin receptor, arealso contemplated as part of the present invention. Antisera thatpossess the highest titer and specificity and can detect angiostatinpeptides in extracts of plasma, urine, tissues, and in cell culturemedia are further examined to establish easy to use kits for rapid,reliable, sensitive, and specific measurement and localization ofangiostatin. These assay kits include but are not limited to thefollowing techniques; competitive and non-competitive assays,radioimmunoassay, bioluminescence and chemiluminescence assays,fluorometric assays, sandwich assays, immunoradiometric assays, dotblots, enzyme linked assays including ELISA, microtiter plates, antibodycoated strips or dipsticks for rapid monitoring of urine or blood, andimmunocytochemistry. For each kit the range, sensitivity, precision,reliability, specificity and reproducibility of the assay areestablished. Intraassay and interassay variation is established at 20%,50% and 80% points on the standard curves of displacement or activity.

One example of an assay kit commonly used in research and in the clinicis a radioimmunoassay (RIA) kit. An angiostatin RIA is illustratedbelow. After successful radioiodination and purification of angiostatinor an angiostatin peptide, the antiserum possessing the highest titer isadded at several dilutions to tubes containing a relatively constantamount of radioactivity, such as 10,000 cpm, in a suitable buffersystem. Other tubes contain buffer or preimmune serum to determine thenon-specific binding. After incubation at 4 C. for 24 hours, protein Ais added and the tubes are vortexed, incubated at room temperature for90 minutes, and centrifuged at approximately 2000-2500 X g at 4° C. toprecipitate the complexes of antibody bound to labeled antigen. Thesupernatant is removed by aspiration and the radioactivity in thepellets counted in a gamma counter. The antiserum dilution that bindsapproximately 10 to 40% of the labeled peptide after subtraction of thenon-specific binding is further characterized.

Next, a dilution range (approximately 0.1 pg to 10 ng) of theangiostatin peptide used for development of the antiserum is evaluatedby adding known amounts of the peptide to tubes containing radiolabeledpeptide and antiserum. After an additional incubation period, forexample, 24 to 48 hours, protein A is added and the tubes centrifuged,supernatant removed and the radioactivity in the pellet counted. Thedisplacement of the binding of radiolabeled angiostatin peptide by theunlabeled angiostatin peptide (standard) provides a standard curve.Several concentrations of other angiostatin peptide fragments,plasminogen, angiostatin from different species, and homologous peptidesare added to the assay tubes to characterize the specificity of theangiostatin antiserum.

Extracts of various tissues, including but not limited to primary andsecondary tumors, Lewis lung carcinoma, cultures of angiostatinproducing cells, placenta, uterus, and other tissues such as brain,liver, and intestine, are prepared using extraction techniques that havebeen successfully employed to extract angiostatin. After lyophilizationor Speed Vac of the tisssue extracts, assay buffer is added anddifferent aliquots are placed into the RIA tubes. Extracts of knownangiostatin producing cells produce displacement curves that areparallel to the standard curve, whereas extracts of tissues that do notproduce angiostatin do not displace radiolabeled angiostatin from theangiostatin antiserum. In addition, extracts of urine, plasma, andcerebrospinal fluid from animals with Lewis lung carcinoma are added tothe assay tubes in increasing amounts. Parallel displacement curvesindicate the utility of the angiostatin assay to measure angiostatin intissues and body fluids.

Tissue extracts that contain angiostatin are additionally characterizedby subjecting aliquots to reverse phase HPLC. Eluate fractions arecollected, dried in Speed Vac, reconstituted in RIA buffer and analyzedin the angiostatin RIA. The maximal amount of angiostatinimmunoreactivity is located in the fractions corresponding to theelution position of angiostatin.

The assay kit provides instructions, antiserum, angiostatin orangiostatin peptide, and possibly radiolabeled angiostatin and/orreagents for precipitation of bound angiostatin-angiostatin antibodycomplexes. The kit is useful for the measurement of angiostatin inbiological fluids and tissue extracts of animals and humans with andwithout tumors.

Another kit is used for localization of angiostatin in tissues andcells. This angiostatin immunohistochemistry kit provides instructions,angiostatin antiserum, and possibly blocking serum and secondaryantiserum linked to a fluorescent molecule such as fluoresceinisothiocyanate, or to some other reagent used to visualize the primaryantiserum. Immunohistochemistry techniques are well known to thoseskilled in the art. This angiostatin immunohistochemistry kit permitslocalization of angiostatin in tissue sections and cultured cells usingboth light and electron microscopy. It is used for both research andclinical purposes. For example, tumors are biopsied or collected andtissue sections cut with a microtome to examine sites of angiostatinproduction. Such information is useful for diagnostic and possiblytherapeutic purposes in the detection and treatment of cancer. Anothermethod to visualize sites of angiostatin biosynthesis involvesradiolabeling nucleic acids for use in in situ hybridization to probefor angiostatin messenger RNA. Similarly, the angiostatin receptor canbe localized, visualized and quantitated with immunohistochemistrytechniques.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLE 1

Choice of an animal-tumor system in which growth of metastasis isinhibited by the primary tumor and is accelerated after removal of theprimary tumor

By screening a variety of murine tumors capable of inhibiting their ownmetastases, a Lewis lung carcinoma was selected in which the primarytumor most efficiently inhibited lung metastasis. Syngeneic C57BI6/Jsix-week-old male mice were injected (subcutaneous dorsum) with 1×10⁶tumor cells. Visible tumors first appeared after 3-4 days. When tumorswere approximately 1500 mm³ in size, mice were randomized into twogroups. The primary tumor was completely excised in the first group andleft intact in the second group after a sham operation. Although tumorsfrom 500 mm³ to 3000 mm³ inhibited growth of metastases, 1500 mm³ wasthe largest primary tumor that could be safely resected with highsurvival and no local recurrence.

After 21 days, all mice were sacrificed and autopsied. In mice with anintact primary tumor, there were four +2 visible metastases, compared tofifty +5 metastases in the mice in which the tumor had been removed(p<0.0001). These data were confirmed by lung weight, which correlatesclosely with tumor burden, as has been previously demonstrated. Therewas a 400% increase in wet lung weight in the mice that had their tumorsremoved compared to mice in which the tumor remained intact (p<0.0001).

This experimental model gave reproducible data and the experimentdescribed is reproducible. This tumor is labeled "Lewis lungcarcinoma---low metastatic" (LLC-Low). The tumor also suppressedmetastases in a nearly identical pattern in SCID mice, which aredeficient in both B and T lymphocytes.

EXAMPLE 2

Isolation of a variant of Lewis lung carcinoma tumor that is highlymetastatic, whether or not the primary tumor is removed

A highly metastatic variant of Lewis lung carcinoma arose spontaneouslyfrom the LLC-Low cell line of Example 1 in one group of mice and hasbeen isolated according to the methods described in Example 1 andrepeatedly transplanted. This tumor (LLC-High) forms more than 30visible lung metastases whether or not the primary tumor is present.

EXAMPLE 3

Size of metastases and proliferation rate of tumor cells within them.Effect of the primary tumor that inhibits metastases (LLC-Low)

C57BI6/J mice were used in all experiments. Mice were inoculatedsubcutaneously with LLC-Low cells, and 14 days later the primary tumorwas removed in half of the mice. At 5, 10 and 15 days after the tumorhad been removed, mice were sacrificed. Histological sections of lungmetastases were obtained. Mice with an intact primary tumor hadmicrometastases in the lung which were not neovascularized. Thesemetastases were restricted to a diameter of 12-15 cell layers and didnot show a significant size increase even 15 days after tumor removal.In contrast, animals from which the primary tumor was removed, revealedlarge vascularized metastases as early as 5 days after operation. Thesemetastases underwent a further 4-fold increase in volume by the 15th dayafter the tumor was removed (as reflected by lung weight and histology).Approximately 50% of the animals who had a primary tumor removed died oflung metastases before the end of the experiment. All animals with anintact primary tumor survived to the end of the experiment.

Replication rate of tumor cells within metastases was determined bycounting nuclei stained with BrdU which had been previously injectedinto the mice. The high percentage of tumor cells incorporating BrdU insmall, avascular metastases of animals with an intact primary tumor wasequivalent to the BrdU incorporation of tumor cells in the largevascularized metastases of mice from which the primary tumor had beenremoved (FIG. 3A). This finding suggests that the presence of a primarytumor has no direct effect on the replication rate of tumor cells withina metastasis.

FIG. 3A shows BrdU labeling index of tumor cells in the lung in thepresence or absence of a primary tumor. Before immunohistochemicalstaining, sections were permeabilized with 0.2 M HCl for 10 minutes anddigested with 1 μg/ml proteinase K (Boehringer Mannheim GmbH, Mannheim,Germany) in 0.2 M Tris-HCl, 2 mM CaCl₂ at 37° C. for 15 minutes.Labeling index was estimated by counting percentage of positive nucleiat 250 power. FIG. 3B depicts an analysis of total lung weight of tumorswith primary tumors intact or removed 5, 10 and 15 days after operation.Animals were sacrificed 6 hours after intraperitoneal injection of BrdU(0.75 mg/mouse).

EXAMPLE 4

Inhibition of angiogenesis in lung metastases in the presence of anintact primary tumor

To measure the degree of vascularization in lung metastases, tissueswere stained with antibodies against von Willebrand factor (anendothelial specific marker, available from Dako Inc., Carpenteria,Calif.). Metastases from animals with intact tumors formed a thin cuff(8-12 tumor cell layers) around existing pulmonary vessels. Except forthe endothelial cells of the vessel lining, no or few cells werepositive for von Willebrand factor. In contrast, lung metastases ofanimals 5 days after removal of the primary tumor were not only largerbut were also infiltrated with capillary sprouts containing endothelialcells which stained strongly for von Willebrand factor.

In immunohistochemical analysis of the presence of endothelial cells inlung metastases, a lung metastasis with the primary lung tumor intact 19days after inoculation, had a cuff of tumor cells around a pre-existingmicrovessel in the lung. The metastasis was limited to 8 to 12 celllayers. There was no evidence of neovascularization around themicrovessel, and it did not contain any new microvessels. This wastypical of the maximum size of an avascular pre-angiogenic metastasis.

In an immunohistochemical analysis of tissue collected five days afterthe primary tumor was resected (19 days after inoculation of the primarytumor), the metastasis surrounded a pre-existing vessel in the lung. Incontrast, in the sample where the primary tumor was not resected, thetumor was neovascularized. Thus, an intact primary tumor inhibitsformation of new capillary blood vessels in metastases, butproliferation of tumor cells within a metastasis are not affected by theprimary tumor.

EXAMPLE 5

A primary tumor inhibits angiogenesis of a second tumor implanted in themouse cornea. Growth of this second tumor is inhibited

A 0.25 to 0.5 mm² Lewis lung tumor (LLC-Low) was implanted in the mousecornea on day 0. (Muthukkaruppan Vr., et al., Angiogenesis in the mousecornea. Science 205:1416-1418, 1979) A primary tumor was formed byinoculating 1×10⁶ LLC-Low cells subcutaneously in the dorsum, either 4or 7 days before the corneal implant; or on the day of the cornealimplant; or 4 or 7 days after the corneal implant. Control mice receivedthe corneal implant but not the subcutaneous tumor. Other control micereceived the corneal implant and an inoculation of LLC-High tumor cellsin the dorsum 4 days before the corneal implant. The corneas wereevaluated daily by slit-lamp stereomicroscopy for the growth of thecorneal tumor (measured by an ocular micrometer) and for the growth ofnew capillary vessels from the edge of the corneal limbus.

In control mice not bearing a primary subcutaneous tumor, a majority ofcorneas (6/8) developed neovascularization starting at day 6 to 7 daysafter corneal implantation and continuing to day 10. By day 10, thevascularized corneal tumors had reached approximately a quarter of thevolume of the whole eye. In the presence of the primary subcutaneousLLC-Low tumor, the corneal implants did not become vascularized if theprimary tumor was in place by at least 4 days or more before the cornealimplant (Table 1). In the absence of neovascularization, corneal tumorsgrew slowly as thin, white, avascular discs within the cornea.

However, if the primary tumor was not implanted until 4 days after thecorneal implant, corneas became vascularized and 3/3 corneal tumors grewat similar rates as the non-tumor bearing controls. In the presence ofthe primary subcutaneous LLC-High tumor, the majority of corneas (2/3)developed neovascularization starting at day 7 after cornealimplantation and continuing to day 10. By day 10, the vascularizedcorneal tumors again had reached approximately a quarter of the volumeof the whole eye.

                  TABLE 1    ______________________________________    Inhibition of tumor angiogenesis in the cornea by    a primary subcutaneous tumor.  All primary tumors are LLC-    Low except (*) which is LLC-High!.    Day of eye implant               0       0      0     0    0     0   0    Day of primary               -7      -4     -4*   0    none  +4  +7    tumor implant    Number of mice               2/10    0/9    2/3   2/3  6/8   3/3 2/3    with new corneal    vessels at day 10    ______________________________________

It would be expected that 0/10 corneas would show neovascularizationwhen the primary LLC-Low subcutaneous tumor was implanted 7 days beforethe eye tumor implant (i.e.-7). However, 2 of the tumors (2/10) hadbecome necrotic because they were too large (>3 cm³).

EXAMPLE 6

Primary intact tumor inhibits angiogenesis induced by a secondarysubcutaneous implant of basic fibroblast growth factor (bFGF.)

Although the experiments described in Examples 4 and 5 show that aprimary tumor inhibits angiogenesis in a secondary metastasis, thesestudies do not reveal whether the primary tumor: (i) inhibitsendothelial proliferation (or angiogenesis) directly, or (ii) indirectlyby down-regulating the angiogenic activity of the metastatic tumorcells. To distinguish between these two possibilities, a focus ofsubcutaneous angiogenesis was induced by an implant of matrigelcontaining basic fibroblast growth factor (bFGF). (Passaniti A, et al.,A simple, quantitative method for assessing angiogenesis andanti-angiogenic agents using reconstituted basement membrane, heparinand fibroblast growth factor. Lab. Invest. 67:519, 1992)

Matrigel (an extract of basement membrane proteins), containing either25 or 50 ng/ml bFGF in the presence of heparin, was injectedsubcutaneously on the ventral surface of normal and tumor-bearing mice(LLC-Low). Mice were sacrificed 4 days later and hemoglobinconcentration in the gel was measured to quantify blood vesselformation. It has previously been shown that the number of new vesselswhich enter the matrigel is correlated with hemoglobin concentration.(Folkman J., Angiogenesis and its inhibitors in "Important Advances inOncology 1985", V T DeVita, S. Hellman and S. Rosenberg, editors, J. B.Lippincott, Philadelphia 1985) Some gels were also prepared forhistological examination. In normal mice, matrigel pellets whichcontained 50 ng/ml bFGF were completely red. They were heavily invadedby new capillary vessels, and contained 2.4 g/dl hemoglobin. Matrigelwhich lacked bFGF was translucent and gray and contained only 0.4 g/dlhemoglobin (a 6-fold difference). In contrast, matrigel from mice with aprimary tumor contained only 0.5 g/dl (FIG. 4).

The near complete inhibition of angiogenesis in this experiment suggeststhat the presence of a Lewis lung primary tumor can inhibit bFGF-inducedangiogenesis directly.

EXAMPLE 7

Transfer of serum from a tumor-bearing animal to an animal from whichthe primary tumor has been removed suppresses metastases

Mice were implanted with Lewis lung carcinoma as described above. After15 days, when tumors were approximately 1500 mm³, the mice wererandomized into four groups. Three groups underwent complete surgicalresection of the primary tumor; in one group the tumors were left inplace (after a sham surgical procedure). The mice in the three resectiongroups then received daily intraperitoneal injections of saline, serumfrom normal nontumor bearing mice, or serum from mice with 1500 mm³Lewis lung carcinomas. The group of mice with the tumors left intactreceived intraperitoneal saline injections. All mice were treated for 21days, after which the animals were euthanized and lung metastases werecounted (Table 2).

                  TABLE 2    ______________________________________                               Primary            Primary Tumor Removed                               Tumor Intact    ______________________________________    Treatment Saline  Serum from Serum from                                         Saline    (Intraperitoneal  normal mice                                 tumor-  Injections    Injections)                  bearing mice    Number of Lung              55 ± 5                      50 ± 4  7 ± 2                                         3 ± 1    Metastases:    ______________________________________

These results were confirmed by lung weight. p=<0.0001 for thedifference between the two groups (55 & 50) vs. (7 & 3)!. Similarresults have been obtained using angiostatin from the urine oftumor-bearing animals.

EXAMPLE 8

Bovine capillary endothelial (BCE) cell assay

BCE cells are used between passages 9 and 14 only. At day 0, BCE cellsare plated onto gelatinized (1.5% gelatin in PBS at 37°, 10% CO₂ for 24hours and then rinsed with 0.5 ml PBS) 24 well plates at a concentrationof 12,500 cells/well. Cell counts are performed using a hemocytometer.Cells are plated in 500 μl DMEM with 10% heat-inactivated (56° C. for 20minutes) bovine calf serum and 1% glutamine-pen-strep (GPS).

BCE cells are challenged as follows: Media is removed and replaced with250 μl of DMEM/5% BCS/1%GPS. The sample to be tested is then added towells. (The amount varies depending on the sample being tested) Platesare placed at 37°C./10% CO₂ for approximately 10 minutes. 250 μl ofDMEM/5% BCS/1% GPS with 2ng/ml bFGF is added to each well. The finalmedia is 500 μl of DMEM/5% BCS1%GPS/ with 1 ng/ml bFGF. The plate isreturned to 37° C./10% CO₂ incubator for 72 hours.

At day 4, cells are counted by removing the medium and then trypsinizingall wells (0.5 ml trypsin/EDTA) for 2 to 3 minutes. The suspended cellsare then transferred to scintillation vials with 9.5 ml Hemetall andcounted using a Coulter counter. A unit of activity is that amount ofserum containing angiostatin that is capable of producing half-maximalinhibition of capillary endothelial proliferation when endothelial cellsare incubated in bFGF 1 ng/ml for 72 hours.

EXAMPLE 9

Serum from mice bearing the low metastatic Lewis lung tumor (LLC-Low)inhibits capillary endothelial cell proliferation in vitro

Bovine capillary endothelial cells were stimulated by basic fibroblastgrowth factor (bFGF 1 ng/ml), in a 72-hour proliferation assay. Theserum of tumor-bearing mice added to these cultures inhibitedendothelial cell proliferation in a dose-dependent and reversiblemanner. Normal serum was not inhibitory (FIG. 5). Endothelial cellproliferation was inhibited in a similar manner (relative to controls)by serum obtained from tumor-bearing nu/nu mice and SCID mice. After theprimary tumor was removed, angiostatin activity disappeared from theserum by 3-5 days.

Tumor-bearing serum also inhibited bovine aortic endothelial cells andendothelial cells derived from a spontaneous mouse hemangioendothelioma,(Obeso, et al., "Methods in Laboratory Investigation, AHemangioendothelioma-derived cell line; Its use as a Model for the Studyof Endothelial Cell Biology," Lab Invest., 63(2), pgs 259-269, 1990) butdid not inhibit Lewis lung tumor cells, 3T3 fibroblasts, aortic smoothmuscle cells, mink lung epithelium, or W138 human fetal lungfibroblasts.

EXAMPLE 10

Serum from mice bearing the Lewis lung tumor (LLC-High) that does notinhibit metastases, does not inhibit capillary endothelial cellproliferation in vitro

Serum from mice bearing a primary tumor of the LLC-High did notsignificantly inhibit proliferation of bFGF-stimulated bovine capillaryendothelial cells relative to controls. Also, when this serum wassubjected to the first two steps of purification (heparin-Sepharosechromatography and gel filtration), angiostatin activity was not foundin any fractions.

EXAMPLE 11

Ascites from Lewis lung carcinoma (low metastatic), also generatesangiostatin serum

Mice received intraperitoneal injections of either LLC-Low or LLC-Hightumor cells (10⁶), and one week later, 1-2 ml of bloody ascites wasobtained from each of 10-20 mice. Mesenteric tumor seeding was seen. Themice were then euthanized. Serum was obtained by cardiac puncture. Serumwas also obtained from normal, non-tumor-bearing mice as a control.Serum and ascites were centrifuged to remove cells, and the supernatewas assayed on bovine capillary endothelial cells stimulated by bFGF (1ng/ml) (see Example 8). Ascites originating from both tumor typesstimulated significant proliferation of capillary endothelial cells(e.g., 100% proliferation) over controls after 72 hours (FIGS. 6A and6B). In contrast, serum from the low metastatic mice inhibitedendothelial cell proliferation (inhibition to 79% of controls) (FIG.6A). The serum from the high metastatic line was stimulatory by 200%(FIG. 6B).

These data show that the ascites of the low metastatic line contains apredominance of endothelial growth stimulator over angiostatin. Thiscondition is analogous to a solid primary tumor. Furthermore,angiostatin activity appears in the serum, as though it were unopposedby stimulatory activity. This pattern is similar to the solid primarytumor (LLC-Low). The ascites from the high metastatic tumor (LLC-High)also appears to contain a predominance of endothelial cell stimulator,but angiostatin cannot be identified in the serum.

EXAMPLE 12

Fractionation of angiostatin from serum by column chromatography andanalysis of growth-inhibitory fractions by SDS-PAGE

To purify the angiostatin(s), serum was pooled from tumor-bearing mice.The inhibitory activity, assayed according the above-described in vitroinhibitor activity assay, was sequentially chromatographed usingheparin-Sepharose, Biogel A0.5 mm agarose, and several cycles ofC4-reverse phase high performance liquid chromatography (HPLC). SDS-PAGEof the HPLC fraction which contained endothelial inhibitory activity,revealed a discrete band of apparent reduced M_(r) Of 38,000 Daltons,which was purified approximately 1 million-fold (see Table 3) to aspecific activity of approximately 2×10⁷. At different stages of thepurification, pooled fractions were tested with specific antibodies forthe presence of known endothelial inhibitors. Platelet factor-4,thrombospondin, or transforming growth factor beta, were not found inthe partially purified or purified fractions.

                  TABLE 3    ______________________________________                Specific activity                (units */mg)                          Fold purification    ______________________________________    Serum          1.69       1    Heparin Sepharose                  14.92       8.8    Bio-gel A0.5m 69.96       41.4    HPLC/C4       2 × 10.sup.7                              1.2 × 10.sup.6    ______________________________________     *A unit of activity is that amount of serum containing angiostatin that i     capable of producing halfmaximal inhibition of capillary endothelial     proliferation when endothelial cells are incubated in bFGF 1 ng/ml for 72     hours.

EXAMPLE 13

Fractionation of angiostatin from urine by column chromatography andanalysis of growth-inhibitory fractions by SDS-PAGE.

Purification of the endothelial cell inhibitor(s) from serum is hamperedby the small volume of serum that can be obtained from each mouse and bythe large amount of protein in the serum.

Urine from tumor bearing mice was analyzed and found that it contains aninhibitor of endothelial cell proliferation that is absent from theurine of non-tumor bearing mice and from mice with LLC-high tumors.Purification of the endothelial cell inhibitory activity was carried outby the same strategy that was employed for purification of serum(described above).

FIGS. 7A and 7B shows C4 reverse phase chromatography of partiallypurified serum or urine from tumor-bearing animals. All fractions wereassayed on bovine capillary endothelial cells with bFGF in a 72-hourproliferation assay as described in Example 8. A discrete peak ofinhibition was seen in both cases eluting at 30-35 % acetonitrile infraction 23. SDS-polyacrylamide gel electrophoresis of inhibitoryfraction from the third cycle of C4 reverse phase chromatography ofserum from tumor-bearing animals showed a single band at about 38,000Daltons.

EXAMPLE 14

Characterization of circulating angiostatin

Endothelial inhibition was assayed according to the procedure describedin Example 9. Angiostatin was isolated on a Synchropak HPLC C4 column.(Synchrom, Inc. Lafayette, Ind.) The inhibitor was eluted at 30 to 35%acetonitrile gradient. On a sodium dodecyl sulfate polyacrylamide gelelectrophoresis (PAGE) gel under reducing conditions(β-mercaptoethanol(5% v/v), the protein band with activityeluted at 38kilodaltons. Under non-reducing conditions, the protein with activityeluted at 28 kilodaltons. The activity is found at similar pointswhether the initial sample was isolated from urine or from serum.Activity was not detected with any other bands.

Activity associated with the bands was lost when heated (100° C. for 10minutes) or treated with trypsin. When the band with activity wasextracted with a water/chloroform mixture (1:1), the activity was foundin the aqueous phase only.

EXAMPLE 15

Purification of inhibitory fragments from human plasminogen

Plasminogen lysine binding site I was obtained from Sigma ChemicalCompany. The preparation is purified human plasminogen after digestionwith elastase. Lysine binding site I obtained in this manner is apopulation of peptides that contain, in aggregate, at least the firstthree triple-loop structures (numbers 1 through 3) in the plasminA-chain (Kringle 1+2+3). (Sotrrup-Jensen, L., et al. in Progress inChemical Fibrinolysis and Thrombolysis, Vol. 3, 191, Davidson, J. F., etal. eds. Raven Press, New York 1978 and Wiman, B., et al., Biochemica etBiophysica Acta, 579, 142 (1979)). Plasminogen lysine binding site I(Sigma Chemical Company, St. Louis, Mo.) was resuspended in water andapplied to a C4-reversed phase column that had been equilibrated withHPLC-grade water/0.1% TFA. The column was eluted with a gradient ofwater/0. 1% TFA to acetonitrile/0.1% TFA and fractions were collectedinto polypropylene tubes. An aliquot of each was evaporated in a speedvac, resuspended with water, and applied to BCEs in a proliferationassay. This procedure was repeated two times for the inhibitoryfractions using a similar gradient for elution. The inhibitory activityeluted at 30-35% acetonitrile in the final run of the C4 column.SDS-PAGE of the inhibitory fraction revealed 3 discrete bands ofapparent reduced molecular mass of 40, 42.5, and 45 kd. SDS-PAGE undernon-reducing conditions revealed three bands of molecular mass 30, 32.5,and 35 kd respectively.

EXAMPLE 16

Extraction of inhibitory activity from SDS-PAGE

Purified inhibitory fractions from human plasminogen based purificationswere resolved by SDS-PAGE under non-denaturing conditions. Areas of thegel corresponding to bands seen in neighboring lanes loaded with thesame samples by silver staining were cut from the gel and incubated in 1ml of phosphate buffered saline at 4° C. for 12 hours in polypropylenetubes. The supernatant was removed and dialyzed twice against saline for6 hours (MWCO=6-8000) and twice against distilled water for 6 hours. Thedialysate was evaporated by vacuum centrifugation. The product wasresuspended in saline and applied to bovine capillary endothelial cellsstimulated by 1 ng/ml basic fibroblast growth factor in a 72 hour assay.Protein extracted from each of the three bands inhibited the capillaryendothelial cells.

EXAMPLE 17

Plasminogen Fragment Treatment Studies

Mice were implanted with Lewis lung carcinomas and underwent resectionswhen the tumors were 1500-2000 mm³. On the day of operation, mice wererandomized into 6 groups of 6 mice each. The mice received dailyintraperitoneal injections with the three purified inhibitory fragmentsof human plasminogen, whole human plasminogen, urine from tumor-bearinganimals, urine from normal mice, or saline. One group of tumor-bearinganimals that had only a sham procedure was treated with salineinjections. Immediately after removal of the primary tumor, the micereceive an intraperitoneal injection of 24 μg (1.2 mg/kg/day/mouse) ofthe inhibitory plasminogen fragments as a loading dose. They thenreceive a daily intraperitoneal injections of 12 μg of the inhibitoryfragment (0.6 mg/kg/day/mouse) for the duration of the experiment.Control mice receive the same dose of the whole plasminogen moleculeafter tumor removal. For the urine treatments, the urine of normal ortumor bearing mice is filtered, dialyzed extensively, lyophilized, andthen resuspended in sterile water to obtain a 250 fold concentration.The mice are given 0.8 ml of the dialyzed urine concentrate, either fromtumor bearing mice or normal mice, in two intraperitoneal injections onthe day of removal of the primary tumor as a loading dose. They thenreceive daily intraperitoneal injections of 0.4 ml of the dialyzed andconcentrated urine for the course of the experiment. Treatments werecontinued for 13 days at which point all mice were sacrificed andautopsied.

The results of the experiment are shown in FIGS. 8 and 9. FIG. 8 showssurface lung metastases after the 13 day treatment. Surface lungmetastases refers to the number of metastases seen in the lungs of themice at autopsy. A stereomicroscope was used to count the metastases.FIG. 8 shows the mean number of surface lung metastases that was countedand the standard error of the mean. As shown, the group of mice with theprimary tumor present showed no metastases. The mice in which theprimary tumor was resected and were treated with saline showed extensivemetastases. The mice treated with the human derived plasminogen fragmentshowed no metastases. The mice treated with whole plasminogen showedextensive metastases indicating that the whole plasminogen molecule hasno endothelial inhibitory activity. Those mice treated with dialyzed andconcentrated urine from tumor bearing mice showed no metastases. Micetreated with concentrated urine from normal mice showed extensivemetastases. When the weight of the lung was measured, similar resultswere obtained (FIG. 9).

EXAMPLE 18

Amino acid sequence of murine and human angiostatin

The amino acid sequence of angiostatin isolated from mouse urine andangiostatin isolated from the human lysine binding site I fragmentpreparation was determined on an Applied Biosystem Model 477A proteinsequencer. Phenylthiohydantoin amino acid fractions were identified withan on-line ABI Model 120A HPLC. The amino acid sequence determined fromthe N-terminal sequence and the tryptic digests of the murine and humanangiostatin indicate that the sequence of the angiostatin is similar tothe sequence beginning at amino acid number 98 of murine plasminogen.Thus, the amino acid sequence of the angiostatin is a moleculecomprising a protein having a molecular weight of between approximately38 kilodaltons and 45 kilodaltons as determined by reducingpolyacrylamide gel electrophoresis and having an amino acid sequencesubstantially similar to that of a murine plasminogen fragment beginningat amino acid number 98 of an intact murine plasminogen molecule. Thebeginning amino acid sequence of the murine angiostatin (SEQ ID NO:2) isshown in FIGS. 1A and 1B. The length of the amino acid sequence may beslightly longer or shorter than that shown in the FIGS. 1A and 1B.

N terminal amino acid analysis and tryptic digests of the activefraction of human lysine binding site I (See Example 15) show that thesequence of the fraction begins at approximately amino acid 97 or 99 ofhuman plasminogen and the human angiostatin is homologous with themurine angiostatin. The beginning amino acid sequence of the humanangiostatin (starting at amino acid 98) is shown in FIGS. 2A, 2B and 2C,(SEQ ID NO:3). The amino acid sequence of murine and human angiostatinis compared in FIGS. 2A, 2B and 2C to corresponding internal amino acidsequences from plasminogen of other species including porcine, bovine,and Rhesus monkey plasminogen, indicating the presence of angiostatin inthose species.

EXAMPLE 19

Expression of human angiostatin in E. coli

The pTrcHisA vector (Invitrogen) (FIG. 10) was used to obtainhigh-level, regulated transcription from the trc promoter for enhancedtranslation efficiency of eukaryotic genes in E. coli. Angiostatin isexpressed fused to an N-terminal nickel-binding poly-histidine tail forone-step purification using metal affinity resins. The enterokinasecleavage recognition site in the fusion peptide allows for subsequentremoval of the N-terminal histidine fusion peptide from the purifiedrecombinant protein. The recombinant human angioststin protein was foundto bind lysine; is cross-reactive with monoclonal antibodies specificfor kringle regions 1, 2 and 3, and inhibits bFGF-driven endothelialcell proliferation in vitro.

To construct the insert, the gene fragment encoding human angiostatin isobtained from human liver mRNA which is reverse transcribed andamplified using the polymerase chain reaction (PCR) and specificprimers. The product of 1131 base pairs encodes amino acids 93 to 470 ofhuman plasminogen. The amplified fragment was cloned into the Xhol/KpnIsite of pTrcHisA, and the resultant construct transformed into XL-1B(available from Stratagene) E. coli host cells. A control clonecontaining the plasmid vector pTrcHisA alone was transformed inot XL-1BE. coli host cells as well. This clone is referred to as the vectorcontrol clone. Both clones were purified identically as described below.

Expressing colonies were selected in the following manner. Colony liftsof E. coli transformed with the gene encoding angiostatin were grown onIPTG impregnated nitrocellulose filters and overlaid on an LB agarplate. Following IPTG induction of expression, colonies were lysed onnitrocellulose filters. The nitrocellulose lifts were blocked, rinsedand probed with two separate monoclonal antibodies (mAbs Dcd and Vap;gift of S. G. McCance and F. J. Castellino, University of Notre Dame)which recognize specific conformations of angiostatin. Stronglyexpressing colonies recognized by the mAbs were selected.

To identify the optimal time for maximal expression, cells werecollected at various times before and after IPTG induction and exposedto repeated freeze-thaw cycles, followed by analysis with SDS-PAGE,immunoblotting and probing with mAbs Dcd and Vap.

From these, clone pTrcHisA/HAsH4 was selected. Induction with IPTG wasfor 4 hours after which the cell pellet was collected and resuspended in50 mM Tris pH 8.0, 2 mM EDTA, 5% glycerol and 200 mg/ml lysozyme andstirred for 30 min. at 4° C. The slurry was centrifuged at 14,000 rpmfor 25 min. and the pellet resuspended in 50 mM Tris pH 8.0, 2 mM EDTA,5% glycerol and 0.1% DOC. This suspension was stirred for 1 hr. at 4°C., and then centrifuged at 14,000 rpm for 25 min. The supernatantfraction at this step contains expressed angiostatin. The E. coliexpressed human angiostatin was found to possess the physical propertyof native angiostatin, that is the ability to bind lysine. The E. coliexpressed angiostatin was thus purified over a lysine-sepharose(Pharmacia or Sigma) column in a single step. Elution of angiostatinfrom the column was with 0.2M epsilon-amino-n-caproic acid pH7.5.

Subsequent to these experiments, scale-up 10 L fermentation batches ofclone pTrcHisA/HAsH4 was performed. The cells obtained from thisscaled-up induction were pelleted and resuspended inSO mM Tris pH7.5,cracked at 10,000 psi thrice chilling at 10° C. in-between passes. Thelysate obtained was clarified by centrifugation at 10,000 rpm for 30 minat 40° C., and expressed angiostatin isolated over lysine-sepharose(FIGS. 11A, 11B and 11C).

Purified E. coli expressed human angiostatin was dialysed exhaustivelyagainst water and lyophilized. The expressed human angiostatin wasresuspended in media (DMEM, 5% BCS, 1% Gentamycin/penicillin/streptomycin) to an estimated concentration of 3 μg/ml, andused in bovine capillary endothelial (BCE) cell assays in vitro, asdescribed in EXAMPLE 8, pg.39. Similarly, the control clone containingthe vector alone was treated in the identical fashion as the clonepTrcHisA/HAsH4. It was induced with IPTG identically, and the bacteriallysate used to bind lysine, eluted with 0.2M amino caproic acid,dialysed exhaustively and lyophilized. This control preparation wasresuspended in media also at an estimated concentration of 3 μg/ml. Thesamples of recombinant angiostatin, and controls were obtained fromdifferent induction and fermentation batches as well as seperatepurification runs, and were all coded at EntreMed, Md. BCE assays wereperformed with these coded samples in a blinded fashion at Children'sHospital, Boston.

The results of BCE assays of recombinant human angiostatin showed thathuman angiostatin expressed in E. coli inhibited the proliferation ofBCE cells due to bFGF (used at 1 ng/ml) (FIG. 12). The stock recombinantangiostatin in media (at about 3 μg/ml) was used at a 1:5, 1:10 and 1:20dilution. Percent inhibition was calculated as follows: ##EQU1## Thepercent inhibition of BCE cell proliferation was comparable or higher tothat of plasminogen derived angiostatin at similar concentrations. Theresults from a repeat run of the BCE assay are depicted in FIG. 13 whereat a 1:5 dilution of the stock, recombinant angiostatin gave similarpercent inhibitions to those obtained with plasminogen derivedangiostatin. FIG. 13 shows the surprising result that human recombinantangiostatin protein inhibits over 60%, and as much as over 75% of BCEproliferation in culture.

EXAMPLE 20

Angiostatin maintains dormancy of micrometastases by increasing the rateof apoptosis

Following subcutaneous inoculation of C57 BL6/J mice with Lewis lungcarcinoma cells (1×10⁶), primary tumors of approximately 1.5 cmdeveloped. Animals were subject to either surgical removal of theprimary tumor or sham surgery. At 5, 10 and 15 days after surgery, micewere sacrificed and their lungs prepared for histological examination.Animals with resected primary tumors showed massive proliferation ofmicrometastases compared to sham operated controls (FIGS. 14A, 14B, 14Cand 14D). These changes were accompanied by a significant increase inlung weight.

Analysis of tumor cell proliferation, as measured by uptake ofbromo-deoxyuridine (BrdU) showed no differences between animals withintact primary tumors or resected tumors at 5, 9 and 13 days, indicatingthat the increase in tumor mass could not be explained by increasedproliferation (FIG. 15). Accordingly, cell death was examined in theseanimals. Apoptosis, a process of cell death that is dependent on changesin gene expression and accounts for elimination of cells duringdevelopment and in rapidly proliferating tissues such as the smallintestine, was examined by immunohistochemically labeling fragmented DNAwith the terminal deoxynucleotidyl transferase (TdT) technique. Theapoptotic index was determined at each time of sacrifice. The removal ofprimary tumors caused a statistically significant increase(approximately 3 to 4 fold) in the apoptotic index at all times examined(FIGS. 15A, 15B and 15C).

Supporting evidence was obtained by treating mice with removed primarytumors with an exogenous suppressor of angiogenesis. This substance,TNP- 1470 (O-chloroacetylcarbamoyl fumagillol, previously namedAGM-1470), is an analogue of fumagillin with reported anti-angiogenicactivity. Subcutaneous injection of TNP-1470 (30 mg/kg every two days)produced results that were strikingly similar to those described abovefor animals that had intact primary tumors. These animals displayed alower lung weight, equivalent proliferative index and increasedapoptotic index compared to saline-injected controls (FIG. 16).

These data indicate that metastases remain dormant when tumor cellproliferation is balanced by an equivalent rate of cell death. Theremoval of the primary tumor causes a rapid increase in the growth ofmetastases, probably due to the removal of angiogenesis inhibitors(angiostatin) which control metastatic growth by increasing apoptosis intumor cells. These effects are similar to those seen following removalof primary tumors and administration of an exogenous inhibitor ofangiogenesis. Taken together, these data suggest that the primary tumorreleases angiostatin which maintains dormancy of micrometastases.

EXAMPLE 21

Treatment of primary tumors with angiostatin in vivo

Angiostatin was purified from human plasminogen by limited elastasedigestion as described in Example 15 above. Angiostatin was resuspendedin phosphate-buffered saline for administration into six week old maleC57BI6/J mice. Animals were implanted subcutaneously with 1×10⁶ tumorcells of either the Lewis lung carcinoma or T241 fibrosarcoma. Treatmentwith angiostatin is begun after four days when tumors are 80-160 mm³ insize. Mice received angiostatin injections in either a single injectionof 40 mg/kg or two 80 mg/kg injections via intraperitoneal (ip) orsubcutaneous (sc) routes. Animals were sacrificed at various times aftertreatment extending to 19 days.

Angiostatin, administered at a daily dose of 40 mg/kg ip, produced ahighly significant inhibition of the growth of T241 primary tumors (FIG.17). This inhibitory effect on growth was visibly evident within 2 daysand increased in magnitude throughout the time course of the study. Byday 18, angiostatin-treated mice had tumors that were approximately 38%of the volume of the saline injected controls. This difference wasstatistically significant (p<0.001, Students t-test).

Angiostatin treatment (total dose of 80 mg/kg/day, administered twicedaily at 40 mg/kg ip or sc) also significantly reduced the growth rateof LLC-LM primary tumors (FIG. 18). This inhibitory effect was evidentat 4 days and increased in magnitude at all subsequent times examined.On the last day of the experiment (day 19), angiostatin-treated micepossessed a mean tumor volume that was only 20% of the saline-injectedcontrols which was significantly different (p<0.001 Students t-test).

In another series of experiments angiostatin was administered (50 mg/kgql2h) to mice implanted with T241 fibrosarcoma, Lewis lung carcinoma(LM) or reticulum cell sarcoma cells. For each tumor cell type, the micereceiving angiostatin had substantially reduced tumor size. FIG. 19demonstrates that for T241 fibrosarcoma, the angiostatin treated micehad mean tumor volumes that were only 15% of the untreated mice at day24. FIG. 20 demonstrates that for Lewis lung carcinoma (LM), theangiostatin treated mice had mean tumor volumes that were only 13% ofthe untreated mice at day 24. FIG. 21 demonstrates that for reticulumsacroma, the angiostatin treated mice had mean tumor volumes that wereonly 19% of the untreated mice at day 24. The data represent the averageof 4 mice at each time point.

These results demonstrate that angiostatin is an extremely potentinhibitor of the growth of three different primary tumors in vivo.

EXAMPLE 22

Treatment of human cell-derived primary tumors in mice with angiostatinin vivo

The effect of angiostatin on two human tumor cell lines, human prostatecarcinoma PC-3 and human breast carcinoma MDA-MB, was studied.Immunodeficient SCID mice were implanted with human tumor cells, and themice treated with 50 mg/kg angiostatin every 12 hours essentially asdescribed in Example 21. The results demonstrate that the angiostatinprotein of the present invention is a potent inhibitor of human tumorcell growth. FIG. 22 shows that for human prostate carcinoma PC-3, theangiostatin treated mice had only 2% of the mean tumor volume comparedto the untreated control mice at day 24. FIG. 23 shows that for humanbreast carcinoma MDA-MB, he angiostatin treated mice had only 8% of themean tumor volume compared to the untreated control mice at day 24.

EXAMPLE 23

Gene Therapy--Effect of transfection of the angiostatin gene on tumorvolume

A 1380 base pair DNA sequence for angiostatin derived from mouseplasminogen cDNA (obtained from American Type Culture Collection(ATCC)), coding for mouse plasminogen amino acids 1-460, was generatedusing PCR and inserted into an expression vector. The expression vectorwas transfected into T241 fibrosarcoma cells and the transfected cellswere implanted into mice. Control mice received either non-transfectedT241 cells, or T241 cells transfected with the vector only (i.e.non-angiostatin expressing transfected cells). Threeangiostatin-expressing transfected cell clones were used in theexperiment. Mean tumor volume determined over time. The results show thesurprising and dramatic reduction in mean tumor volume in mice for theangiostatin-expressing cells clones as compared with the non-transfectedand non-expressing control cells.

The mouse DNA sequence coding for mouse angiostatin protein is derivedfrom mouse plasminogen cDNA. The mouse angiostatin encompasses mouseplasminogen kringle regions 1-4. The schematic for constructing thisclone is shown in FIG. 24.

The mouse angiostatin protein clones were transfected into T241fibrosarcoma cells using the LIPOFECTIN™ transfection system (availablefrom Life Technologies, Gaithersburg, Md.). The LIPOFECTIN™ reagent is a1:1 (w/w) liposome formulation of the cationic lipid N-1-(2,3-dioleyloxy)propyl!-n,n,n-trimethylammonium chloride (DOTMA), anddiolecoyl phosphotidylethanolamine (DOPE) in membrane filtered water.

The procedure for transient transfection of cells is as follows:

1. T241 cells are grown in 60 cm² tissue culture dishes, seed≈1-10⁵cells in 2 ml of the appropriate growth medium supplemented with serum.

2. Incubate the cells at 37° C. in a CO₂ incubator until the cells are40-70% confluent. will usually take 18-24 h, but the time will varyamong cell types. The T241 tumor cells confluency was approximately 70%.

3. Prepare the following solutions in 12×75 mm sterile tubes:

Solution A: For each transfection, dilute 5 μg of DNA in 100 μl ofserum-free OPTI-MEM I Reduced Serum Medium (available from LifeTechnologies) (tissue culture grade deionized water can also be used).

Solution B: For each transfection, dilute 30 μg of LIPOFECTIN in 100 μlOPTI-MEM medium.

4. Combine the two solutions, mix gently, and incubate at roomtemperature for 10-15 min.

5. Wash cells twice with serum-free medium.

6. For each transfection, add 0.8 ml serum-free medium to each tubecontaining the LIPOFECTIN™ reagent-DNA complexes. Mix gently and overlaythe complex onto cells.

7. Incubate the cells for approximately 12 h at 37° C. in a CO₂incubator.

8. Replace the DNA containing medium with 1 mg/ml selection mediumcontaining serum and incubate cells at 37° C. in a CO₂ incubator for atotal of 48-72 h.

9. Assay cell extracts for gene activity at 48-72 h post transfection.

Transfected cells can be assayed for expression of angiostatin proteinusing angiostatin-specific antibodies. Alternatively, after about 10-14days, G418 resistant colonies appeared in the CMV-angiostatintransfected T241 cells. Also, a number of clones were seen in the vectoralone transfected clones but not in the untransfected clones. The G418resistant clones were selected for their expression of angiostatin,using a immunofluorence method.

Interestingly, the in vitro cell growth T241 cells and Lewis lung cellstransfected with angiostatin was not inhibited or otherwise adverselyaffected, as shown in FIGS. 25A, 25B, 26A, 26B and 26C.

FIG. 27 depicts the results of the transfection experiment. All three ofthe angiostatin-expressing T241 transfected clones produced mean tumorvolumes in mice that were substantially reduced relative to the tumorvolume in contol mice. The mean tumor volume of the mice implanted withClone 37 was only 13% of the control, while Clone 31 and Clone 25 tumorvolumes were only 21% and 34% of the control tumor volumes,respectively. These results demonstrate that the DNA sequences codingfor angiostatin can be transfected into cells, that the transfected DNAsequences are capable of expressing angiostatin protein by implantedcells, and that the expressed angiostatin fucntions in vivo to reducetumor growth.

EXAMPLE 24

Localization of in vivo site of angiostatin expression

To localize the in vivo site of expression of angiostatin protein, totalRNA from various cell types, Lewis lung carcinoma cells (mouse), T241fibrosarcoma (mouse), and Burkitt's lymphoma cells (human), both fromfresh tumor or cell culture after several passages were analysed todetermine the presence of angiostatin transcripts. Northern analysis ofsamples showed an absence of any signal hybridizing with thn sequencefrom all samples except that of normal mouse liver RNA showing a singlesignal of approximately 2.4 kb corresponding to mouse plasminogen.Northern analysis of human samles show an absence of any signalhybridizing with human angiostatin sequence from all samples except thatof normal human liver RNA showing a single signal of approximately 2.4kb corresponding to human plasminogen.

Reverse transcription polymerase chain reaction (RT-PCR) analysis showedan absence of any product from all samples probed with mouse angiostatinsequences except that of the normal mouse liver. RT-PCR analysis showedan absence of any product from all human samples probed with humanangiostatin sequences except that of the normal human liver (expectedsize of 1050 bp for mouse and 1134 bp for human).

Thus it appears that mouse angiostatin transcripts (assuming identitywith amino acids 97 to 450 of mouse plasminogen) are not produced by allthe above mouse samples and human angiostatin transcripts (assumingidentity with amino acids 93 to 470 of human plasminogen) are notproduced by the above human samples. The positive signals obtained innormal mouse/human liver is from hybridization with plasminogen.

EXAMPLE 25

Expression of Angiostatin in Yeast

The gene fragment encoding amino acids 93 to 470 of human plasminogenwas cloned into the XhoI/EcoRI site of pHIL-SI(Invitrogen) which allowsthe secreted expression of proteins using the PHO1 secretion signal inthe yeast Pichia pastoris. Similarly, the gene fragment encoding aminoacids 93 to 470 of human plasminogen was cloned into the SnaBI/EcoRIsite of pPIC9 (Invitrogen) which allows the secreted expression ofproteins using the α-factor secretion signal in the yeast Pichiapastoris. The expressed human angiostatin proteins in these systems willhave many advantages over those expressed in E. coli such as proteinprocessing, protein folding and posttranslational modification inclusiveof glycosylation.

Expression of gene in P. pastoris: is described in) Sreekrishna, K. etal. (1988) High level expression of heterologous proteins inmethylotropic yeast Pichia pastoris. J. Basic Microbiol. 29 (4):265-278, and Clare, J. J. et al. (1991) Production of epidermal growthfactor in yeast: High-level secretion using Pichia pastoris strainscontaining multiple gene copies, Gene 105:205-212, both of which arehereby incorporated herein by reference.

EXAMPLE 26

Expression of angiostatin proteins in transgenic animals and plants

Transgenic animals such as of the bovine or procine family are createdwhich express the angiostatin gene transcript. The transgenic animalexpress angiostatin protein for example in the milk of these animals.Additionally edible transgenic plants which express the angiostatin genetranscript are constructed.

Constructing transgenic animals that express foreign DNA is described inSmith H. Phytochrome transgenics: functional, ecological andbiotechnical applications, Semin. Cell. Biol. 1994 5(5):315-325, whichis hereby incorporated herein by reference.

It should be understood that the foregoing relates only to preferredembodiments of the present invention, and that numerous modifications oralterations may be made therein without departing from the spirit andthe scope of the invention as set forth in the appended claims.

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GluGlnThrProValValGlnGluCysTyrGlnSerAspGlyGlnSer    370375380    TyrArgGlyThrSerSerThrThrIleThrGlyLysLysCysGlnSer    385390395400    TrpAlaAlaMetPheProHisArgHisSerLysThrProGluAsnPhe    405410415    ProAspAlaGlyLeuGluMetAsnTyrCysArgAsnProAspGlyAsp    420425430    LysGlyProTrpCysTyrThrThrAspProSerValArgTrpGluTyr    435440445    CysAsnLeuLysArgCysSerGluThrGlyGlySerValValGluLeu    450455460    ProThrValSerGlnGluProSerGlyProSerAspSerGluThrAsp    465470475480    CysMetTyrGlyAsnGlyLysAspTyrArgGlyLysThrAlaValThr    485490495    AlaAlaGlyThrProCysGlnGlyTrpAlaAlaGlnGluProHisArg    500505510    HisSerIlePheThrProGlnThrAsnProArgAlaAspLeuGluLys    515520525    AsnTyrCysArgAsnProAspGlyAspValAsnGlyProTrpCysTyr    530535540    ThrThrAsnProArgLysLeuTyrAspTyrCysAspIleProLeuCys    545550555560    AlaSerAlaSerSerPheGluCysGlyLysProGlnValGluProLys    565570575    LysCysProGlyArgValValGlyGlyCysValAlaAsnProHisSer    580585590    TrpProTrpGlnIleSerLeuArgThrArgPheThrGlyGlnHisPhe    595600605    CysGlyGlyThrLeuIleAlaProGluTrpValLeuThrAlaAlaHis    610615620    CysLeuGluLysSerSerArgProGluPheTyrLysValIleLeuGly    625630635640    AlaHisGluGluTyrIleArgGlyLeuAspValGlnGluIleSerVal    645650655    AlaLysLeuIleLeuGluProAsnAsnArgAspIleAlaLeuLeuLys    660665670    LeuSerArgProAlaThrIleThrAspLysValIleProAlaCysLeu    675680685    ProSerProAsnTyrMetValAlaAspArgThrIleCysTyrIleThr    690695700    GlyTrpGlyGluThrGlnGlyThrPheGlyAlaGlyArgLeuLysGlu    705710715720    AlaGlnLeuProValIleGluAsnLysValCysAsnArgValGluTyr    725730735    LeuAsnAsnArgValLysSerThrGluLeuCysAlaGlyGlnLeuAla    740745750    GlyGlyValAspSerCysGlnGlyAspSerGlyGlyProLeuValCys    755760765    PheGluLysAspLysTyrIleLeuGlnGlyValThrSerTrpGlyLeu    770775780    GlyCysAlaArgProAsnLysProGlyValTyrValArgValSerArg    785790795800    PheValAspTrpIleGluArgGluMetArgAsnAsn    805810    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 339 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Murine    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    ValTyrLeuSerGluCysLysThrGlyIleGlyAsnGlyTyrArgGly    151015    ThrMetSerArgThrLysSerGlyValAlaCysGlnLysTrpGlyAla    202530    ThrPheProHisValProAsnTyrSerProSerThrHisProAsnGlu    354045    GlyLeuGluGluAsnTyrCysArgAsnProAspAsnAspGluGlnGly    505560    ProTrpCysTyrThrThrAspProAspLysArgTyrAspTyrCysAsn    65707580    IleProGluCysGluGluGluCysMetTyrCysSerGlyGluLysTyr    859095    GluGlyLysIleSerLysThrMetSerGlyLeuAspCysGlnAlaTrp    100105110    AspSerGlnSerProHisAlaHisGlyTyrIleProAlaLysPhePro    115120125    SerLysAsnLeuLysMetAsnTyrCysHisAsnProAspGlyGluPro    130135140    ArgProTrpCysPheThrThrAspProThrLysArgTrpGluTyrCys    145150155160    AspIleProArgCysThrThrProProProProProSerProThrTyr    165170175    GlnCysLeuLysGlyArgGlyGluAsnTyrArgGlyThrValSerVal    180185190    ThrValSerGlyLysThrCysGlnArgTrpSerGluGlnThrProHis    195200205    ArgHisAsnArgThrProGluAsnPheProCysLysAsnLeuGluGlu    210215220    AsnTyrCysArgAsnProAspGlyGluThrAlaProTrpCysTyrThr    225230235240    ThrAspSerGlnLeuArgTrpGluTyrCysGluIleProSerCysGlu    245250255    SerSerAlaSerProAspGlnSerAspSerSerValProProGluGlu    260265270    GlnThrProValValGlnGluCysTyrGlnSerAspGlyGlnSerTyr    275280285    ArgGlyThrSerSerThrThrIleThrGlyLysLysCysGlnSerTrp    290295300    AlaAlaMetPheProHisArgHisSerLysThrProGluAsnPhePro    305310315320    AspAlaGlyLeuGluMetAsnTyrCysArgAsnProAspGlyAspLys    325330335    GlyProTrp    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 339 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Homo sapiens    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    ValTyrLeuSerGluCysLysThrGlyAsnGlyLysAsnTyrArgGly    151015    ThrMetSerLysThrLysAsnGlyIleThrCysGlnLysTrpSerSer    202530    ThrSerProHisArgProArgPheSerProAlaThrHisProSerGlu    354045    GlyLeuGluGluAsnTyrCysArgAsnProAspAsnAspProGlnGly    505560    ProTrpCysTyrThrThrAspProGluLysArgTyrAspTyrCysAsp    65707580    IleLeuGluCysGluGluGluCysMetHisCysSerGlyGluAsnTyr    859095    AspGlyLysIleSerLysThrMetSerGlyLeuGluCysGlnAlaTrp    100105110    AspSerGlnSerProHisAlaHisGlyTyrIleProSerLysPhePro    115120125    AsnLysAsnLeuLysLysAsnTyrCysArgAsnProAspArgGluLeu    130135140    ArgProTrpCysPheThrThrAspProAsnLysArgTrpGluLeuCys    145150155160    AspIleProArgCysThrThrProProProSerSerGlyProThrTyr    165170175    GlnCysLeuLysGlyThrGlyGluAsnTyrArgGlyAsnValAlaVal    180185190    ThrValSerGlyHisThrCysGlnHisTrpSerAlaGlnThrProHis    195200205    ThrHisAsnArgThrProGluAsnPheProCysLysAsnLeuAspGlu    210215220    AsnTyrCysArgAsnProAspGlyLysArgAlaProTrpCysHisThr    225230235240    ThrAsnSerGlnValArgTrpGluTyrCysLysIleProSerCysAsp    245250255    SerSerProValSerThrGluGlnLeuAlaProThrAlaProProGlu    260265270    LeuThrProValValGlnAspCysTyrHisGlyAspGlyGlnSerTyr    275280285    ArgGlyThrSerSerThrThrThrThrGlyLysLysCysGlnSerTrp    290295300    SerSerMetThrProHisArgHisGlnLysThrProGluAsnTyrPro    305310315320    AsnAlaGlyLeuThrMetAsnTyrCysArgAsnProAspAlaAspLys    325330335    GlyProTrp    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 339 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Rhesus monkey    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    ValTyrLeuSerGluCysLysThrGlyAsnGlyLysAsnTyrArgGly    151015    ThrMetSerLysThrArgThrGlyIleThrCysGlnLysTrpSerSer    202530    ThrSerProHisArgProThrPheSerProAlaThrHisProSerGlu    354045    GlyLeuGluGluAsnTyrCysArgAsnProAspAsnAspGlyGlnGly    505560    ProTrpCysTyrThrThrAspProGluGluArgPheAspTyrCysAsp    65707580    IleProGluCysGluAspGluCysMetHisCysSerGlyGluAsnTyr    859095    AspGlyLysIleSerLysThrMetSerGlyLeuGluCysGlnAlaTrp    100105110    AspSerGlnSerProHisAlaHisGlyTyrIleProSerLysPhePro    115120125    AsnLysAsnLeuLysLysAsnTyrCysArgAsnProAspGlyGluPro    130135140    ArgProTrpCysPheThrThrAspProAsnLysArgTrpGluLeuCys    145150155160    AspIleProArgCysThrThrProProProSerSerGlyProThrTyr    165170175    GlnCysLeuLysGlyThrGlyGluAsnTyrArgGlyAspValAlaVal    180185190    ThrValSerGlyHisThrCysHisGlyTrpSerAlaGlnThrProHis    195200205    ThrHisAsnArgThrProGluAsnPheProCysLysAsnLeuAspGlu    210215220    AsnTyrCysArgAsnProAspGlyGluLysAlaProTrpCysTyrThr    225230235240    ThrAsnSerGlnValArgTrpGluTyrCysLysIleProSerCysGlu    245250255    SerSerProValSerThrGluProLeuAspProThrAlaProProGlu    260265270    LeuThrProValValGlnGluCysTyrHisGlyAspGlyGlnSerTyr    275280285    ArgGlyThrSerSerThrThrThrThrGlyLysLysCysGlnSerTrp    290295300    SerSerMetThrProHisTrpHisGluLysThrProGluAsnPhePro    305310315320    AsnAlaGlyLeuThrMetAsnTyrCysArgAsnProAspAlaAspLys    325330335    GlyProTrp    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 339 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Porcine    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    IleTyrLeuSerGluCysLysThrGlyAsnGlyLysAsnTyrArgGly    151015    ThrThrSerLysThrLysSerGlyValIleCysGlnLysTrpSerVal    202530    SerSerProHisIleProLysTyrSerProGluLysPheProLeuAla    354045    GlyLeuGluGluAsnTyrCysArgAsnProAspAsnAspGluLysGly    505560    ProTrpCysTyrThrThrAspProGluThrArgPheAspTyrCysAsp    65707580    IleProGluCysGluAspGluCysMetHisCysSerGlyGluHisTyr    859095    GluGlyLysIleSerLysThrMetSerGlyIleGluCysGlnSerTrp    100105110    GlySerGlnSerProHisAlaHisGlyTyrLeuProSerLysPhePro    115120125    AsnLysAsnLeuLysMetAsnTyrCysArgAsnProAspGlyGluPro    130135140    ArgProTrpCysPheThrThrAspProAsnLysArgTrpGluPheCys    145150155160    AspIleProArgCysThrThrProProProThrSerGlyProThrTyr    165170175    GlnCysLeuLysGlyArgGlyGluAsnTyrArgGlyThrValSerVal    180185190    ThrAlaSerGlyHisThrCysGlnArgTrpSerAlaGlnSerProHis    195200205    LysHisAsnArgThrProGluAsnPheProCysLysAsnLeuGluGlu    210215220    AsnTyrCysArgAsnProAspGlyGluThrAlaProTrpCysTyrThr    225230235240    ThrAspSerGluValArgTrpAspTyrCysLysIleProSerCysGly    245250255    SerSerThrThrSerThrGluHisLeuAspAlaProValProProGlu    260265270    GlnThrProValAlaGlnAspCysTyrArgGlyAsnGlyGluSerTyr    275280285    ArgGlyThrSerSerThrThrIleThrGlyArgLysCysGlnSerTrp    290295300    ValSerMetThrProHisArgHisGluLysThrProGlyAsnPhePro    305310315320    AsnAlaGlyLeuThrMetAsnTyrCysArgAsnProAspAlaAspLys    325330335    SerProTrp    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 339 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Bovine    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    IleTyrLeuLeuGluCysLysThrGlyAsnGlyGlnThrTyrArgGly    151015    ThrThrAlaGluThrLysSerGlyValThrCysGlnLysTrpSerAla    202530    ThrSerProHisValProLysPheSerProGluLysPheProLeuAla    354045    GlyLeuGluGluAsnTyrCysArgAsnProAspAsnAspGluAsnGly    505560    ProTrpCysTyrThrThrAspProAspLysArgTyrAspTyrCysAsp    65707580    IleProGluCysGluAspLysCysMetHisCysSerGlyGluAsnTyr    859095    GluGlyLysIleAlaLysThrMetSerGlyArgAspCysGlnAlaTrp    100105110    AspSerGlnSerProHisAlaHisGlyTyrIleProSerLysPhePro    115120125    AsnLysAsnLeuLysMetAsnTyrCysArgAsnProAspGlyGluPro    130135140    ArgProTrpCysPheThrThrAspProGlnLysArgTrpGluPheCys    145150155160    AspIleProArgCysThrThrProProProSerSerGlyProLysTyr    165170175    GlnCysLeuLysGlyThrGlyLysAsnTyrGlyGlyThrValAlaVal    180185190    ThrGluSerGlyHisThrCysGlnArgTrpSerGluGlnThrProHis    195200205    LysHisAsnArgThrProGluAsnPheProCysLysAsnLeuGluGlu    210215220    AsnTyrCysArgAsnProAspGlyGluLysAlaProTrpCysTyrThr    225230235240    ThrAsnSerGluValArgTrpGluTyrCysThrIleProSerCysGlu    245250255    SerSerProLeuSerThrGluArgMetAspValProValProProGlu    260265270    GlnThrProValProGlnAspCysTyrHisGlyAsnGlyGlnSerTyr    275280285    ArgGlyThrSerSerThrThrIleThrGlyArgLysCysGlnSerTrp    290295300    SerSerMetThrProHisArgHisLeuLysThrProGluAsnTyrPro    305310315320    AsnAlaGlyLeuThrMetAsnTyrCysArgAsnProAspAlaAspLys    325330335    SerProTrp    __________________________________________________________________________

We claim:
 1. A method of expressing in vitro an angiostatin proteincomprising, transfecting a vector into a cell, wherein the vectorcomprises a nucleotide molecule have a sequence that encodes for anangiostatin protein containing approximately kringle regions 1 trough 4of a plasminogen molecule, the angiostatin protein having ananti-angiogenic activity and having a molecular weight of betweenapproximately 38 and 45 kilodaltons as determined by reducingpolyacrylamide gel electrophoresis, wherein the vector is capable ofexpressing the angiostatin protein when present in the cell.
 2. Themethod of claim 1, wherein the nucleotide sequence encodes for abeginning amino acid sequence selected from the group consisting of SEQID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:
 6. 3.The method of claim 1, wherein the nucleotide sequence encodes for anangiostatin protein beginning at approximately amino acid 98 of aplasminogen molecule.
 4. The method of claim 1, wherein the nucleotideencodes for an angiostatin protein capable of inhibiting the growth ofcancer.
 5. The method of claim 4, wherein the cancer is selected fromthe group consisting of breast cancer, colon cancer, lung cancer,prostate cancer, fibroblast cancer and reticulum cancer.
 6. A method ofexpressing in vitro an angiostatin protein comprising, transfecting intoa cell a vector, wherein the vector contains a nucleotide moleculehaving a sequence that encodes for an angiostatin protein having ananti-angiogenic activity and containing approximately kringle regions 1through 4 of a plasminogen molecule wherein the cell is capable ofexpressing the angiostatin protein.
 7. The method of claim 6, whereinthe nucleotide sequence encodes for a beginning amino acid sequenceselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, and SEQ ID NO:
 6. 8. The method of claim 6 whereinthe nucleotide sequence encodes for an angiostatin protein beginning atapproximately amino acid 98 of a plasminogen molecule.
 9. The method ofclaim 6 wherein the nucleotide encodes for an angiostatin proteincapable of inhibiting the growth of a cancer.
 10. The method of claim 9,wherein the cancer is selected from the group consisting of breastcancer, colon cancer, lung cancer, prostate cancer, fibroblast cancerand reticulum cancer.
 11. A method of expressing in vitro an angiostatinprotein comprising, transfecting a vector into a cell, wherein thevector contains a nucleotide molecule having a sequence that encodes foran angiostatin protein having an anti-angiogenic activity, having amolecular weight of between approximately 38 and 45 kilodaltons asdetermined by reducing polyacrylamide gel electrophoresis, and having anamino acid sequence substantially similar to a plasminogen fragmentbeginning at about amino acid 98 of a plasminogen molecule.
 12. Themethod of claim 11, wherein the angiostatin protein codes for abeginning amino acid sequence selected from the group consisting of SEQID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:
 6. 13.The method of claim 11, wherein the nucleotide sequence encodes for abeginning amino acid sequence substantially similar to SEQ ID NO:2. 14.The method of claim 11, wherein the angiostatin protein has in vitroendothelial cell proliferation inhibiting activity.
 15. The method ofclaim 11, wherein the angiostatin protein is capable of inhibiting thegrowth of cancer.
 16. The method of claim 15, wherein the cancer isselected from the group consisting of breast cancer, colon cancer, lungcancer, prostate cancer, fibroblast cancer and reticulum cancer.
 17. Themethod of claim 1, wherein the nucleotide sequence encodes for abeginning amino acid sequence substantially similar to SEQ ID NO:2. 18.The method of claim 1, wherein the angiostatin protein has in vitroendothelial cell proliferation inhibiting activity.
 19. The method ofclaim 6, wherein the nucleotide sequence encodes for a beginning aminoacid sequence substantially similar to SEQ ID NO:2.
 20. The method ofclaim 6, wherein the angiostatin protein has in vitro endothelial cellproliferation inhibiting activity.